Method for detection of PF4A receptor nucleic acid

ABSTRACT

cDNAs encoding a class of receptors, including the IL-8 receptors, have been identified in human tissue. Recombinantly produced PF4ARs are used in the preparation and purification of antibodies capable of binding to the receptors, and in diagnostic assays. The antibodies are advantageously used in the prevention and treatment of inflammatory conditions. The cDNAs are also used to detect nucleic acids in samples. The cDNAs are also used to detect nucleic acids in samples.

This application is a divisional application of U.S. Ser. No. 08/284,586filed Aug. 10, 1994, now issued as U.S. Pat. No. 5,840,856 which is a 35U.S.C. §371 application of International Application No. PCT/US94/06380filed Jun. 7, 1994, now inactive, which is a continuation of U.S. Ser.No. 08/076,093, filed Jun. 11, 1993 and now issued as U.S. Pat. No.5,543,503, which is a continuation of U.S. Ser. No. 07/810,782 filedDec. 19, 1991, now abandoned, which is a continuation-in-partapplication of U.S. Ser. No. 07/677,211 filed Mar. 29, 1991, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of assaying platelet factor 4superfamily members (hereafter "PF4A") and the preparation of agonistsand antagonists to the members of this family, in particular, antibodiesto these receptors.

2. Description of Background and Related Art

While interleukin-8 was initially identified as a chemoattractant forneutrophils, and was known to bind a receptor on neutrophils (Samanta etal., J. Exp. Med., 169: 1185-1189 1989!; Besemer et al., J. Biol. Chem.,264: 17409-17415 1989!; Grob et al. J. Biol. Chem., 265: 8311-83161990!), it has in addition a wide range of pro-inflammatory activitiesincluding the stimulation of degranulation and the upregulation of thecell adhesion molecule MAC-1 and of the complement receptor CR1.Oppenheim et al., Annu. Rev. Immunol., 9: 617-648 (1991).

IL-8 is secreted by many cell types in response to pro-inflammatorystimuli such as IL-1β, TNF, and endotoxin and is a member of a family often or more pro-inflammatorycytokines with an M_(r) ˜10,000. Oppenheimet al., supra. This larger family of proteins is called the plateletfactor 4 superfamily. Wolpe et al., FASEB J., 3: 2565-73 (1989). Somemembers of the platelet factor 4 superfamily, in general the subsetreferred to as C-X-C peptides (including IL-8), possess neutrophilagonist activity, e.g. neutrophil activating protein-2 (NAP-2), plateletfactor 4 and NAP-3 (melanoma growth-stimulating activity MGSA!/gro), allof which are encoded by genes on human chromosome 4. Other members ofthis family, the C-C peptides, encoded by genes on human chromosome 17,are not neutrophil agonists, and include RANTES, macrophage chemotacticand activating factor (MCAF). Hereafter "PF4A" means the PF4superfamily. Oppenheim et al., supra.

The IL-8 receptors are members of the superfamily of seventransmembrane, G-protein linked receptors. Taylor, Biochem. J., 272: 1(1990). This family of receptors includes several hundred differentreceptors among which the β-adrenergic receptor (Strader et al., FASEB,3: 1825 1989!; Dixon et al., EMBO J., 6: 3269 1987!), the muscarinic andcholinergic receptors (Kubo et al., Nature, 323: 411 1986!; Peralta etal., EMBO J., 6: 3923 1987! ), the c5a and fMet-Leu-Phe receptors. Twotypes of IL-8 receptors have been described: type A (IL8R-A) (Holmes etal., Science, 253: 1278 1991!) and type B (IL8R-B) (Murphy and Tiffany,Science, 253: 1280 1991!) receptors. These two receptors share 77% aminoacid identity and have 29-34% sequence homology to C5a and fMet-Leu-Phe.Holmes et al., supra. IL8R-A has a high affinity (2 nM) for IL-8 only,while IL8R-B has a high affinity (2 nM) for both IL-8 and MGSA. Thefunction and expression level of each receptor on these cells have yetto be determined.

It is an object of this invention to identify receptors for the PF4Asuperfamily (hereinafter "PF4AR").

It is another object of this invention to obtain DNA encoding orhybridizing to these receptors, and to express the receptors in hostcells.

It is an additional object of this invention to provide isolates ofPF4AR for diagnostic and therapeutic purposes.

A still further object is to obtain DNA encoding variants of suchreceptors and to prepare such variants in recombinant cell culture.

A yet further object is to identify and prepare antibodies to receptorsfor the PF4A superfamily (hereinafter "PF4AR").

It is still another object to provide a method for treating orpreventing an inflammatory response in a mammal using an antibody tosuch receptors.

These and other objects of this invention will be apparent from thespecification as a whole.

SUMMARY OF THE INVENTION

These objects are accomplished, in one aspect, by providing an isolatednovel PF4AR polypeptide, including polypeptides that are relatedstructurally to the PF4AR. Members of this class of polypeptide arehereafter generically termed PF4AR, and include derivatives and variantsthereof.

Either the whole PF4AR molecule or fragments thereof (which also may besynthesized by chemical methods) fused (by recombinant expression or invitro covalent methods) to an immunogenic polypeptide are used toimmunize an animal to raise antibodies against a PF4AR epitope.Anti-PF4AR antibodies are recovered from the serum of immunized animals.Alternatively, monoclonal antibodies are prepared from cells of theimmunized animal in conventional fashion.

Thus, in a further aspect, the invention provides an antibody that iscapable of binding a PF4AR polypeptide, preferably one that does notcross-react with a receptor capable of binding another PF4 superfamilymember. More preferably, the antibody is capable of binding an IL-8receptor, most preferably an IL-8 type A receptor. Also, the preferredantibody has the isotype IgG1 and/or neutralizes the in vitro activityof a PF4AR polypeptide, preferably of an IL-8 type A receptor.

Specific preferred antibodies herein are the monoclonal antibodiesdesignated 2A4, having ATCC Deposit No. HB 11377, and 9H1, having ATCCDeposit No. HB 11376.

Anti-PF4AR antibodies are useful particularly in the diagnosis (in vitroor in vivo) or (when immobilized on an insoluble matrix) thepurification of the PF4AR. The antibodies are also useful in treatmentof an inflammatory response in patients. Thus, in another aspect, theinvention provides a composition comprising the antibody and apharmaceutically acceptable carrier, as well as a method for treating aninflammatory disorder which method comprises administering to a mammalin need of such treatment an effective amount of this composition.

Substitutional, deletional, or insertional variants of the PF4AR areprepared by in vitro or recombinant methods and screened forimmuno-crossreactivity with the PF4AR and for PF4AR antagonist oragonist activity.

The PF4AR also is derivatized in vitro to prepare immobilized PF4AR andlabeled PF4AR, particularly for purposes of diagnosis of PF4AR or itsantibodies, or for affinity purification of PF4AR antibodies.

The PF4AR, its derivatives, or its antibodies are formulated intophysiologically acceptable vehicles, especially for therapeutic use.Such vehicles include sustained-releaseformulations of the PF4AR.

In still other aspects, the invention provides an isolated nucleic acidmolecule encoding the PF4AR, labeled or unlabeled, and a nucleic acidsequence that is complementary to, or hybridizes under suitableconditions to a nucleic acid sequence encoding the PF4AR.

In addition, the invention provides a replicable vector comprising thenucleic acid molecule encoding the PF4AR operably linked to controlsequences recognized by a host transformed by the vector; host cellstransformed with the vector; and a method of using a nucleic acidmolecule encoding the PF4AR to effect the production of PF4AR,comprising expressing the nucleic acid molecule in a culture of thetransformed host cells and recovering the PF4AR from the host cellculture. The nucleic acid sequence is also useful in hybridizationassays for PF4AR nucleic acid. The recombinant host cells areparticularly useful in assaying the appropriate PF4A members.

In further embodiments, the invention provides a method for producingPF4AR comprising inserting into the DNA of a cell containing the nucleicacid encoding the PF4AR a transcription modulatory element in sufficientproximity and orientation to the PF4AR nucleic acid to influence ordestroy transcription of DNA encoding a biologically active PF4AR, withan optional further step comprising culturing the cell containing thetranscription modulatory element and the PF4AR nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the high affinity binding of IL-8 to COS cellstransfected with clone pRK5B.il8r1.1. a, Competition with unlabelledIL-8 or fMLP. b, Scatchard analysis of the IL-8 competition data;apparent Kd=3.6 nM, average of 820,000 binding sites/cell. Similarcompetitions with human neutrophils gave Kd=1.1 nM, 31000 bindingsites/cell.

FIGS. 2a-2c (hereinafter referred to collectively as FIG. 2) depict theamino acid (SEQ ID NO. 2) and nucleotide (SEQ ID NO, 1) sequences of theIL-8 receptor cDNA insert from clone pRK5B.il8r1.1. The seven putativetransmembrane domains are shown. There are 4 extracellular segments and4 intracellular segments, each being separated by one of thetransmembrane domains. The extracellular segments are approximatelydelineated by residues 1-39, 99-111, 134-154, 175-203 and 265-290. TheIL-8 receptor contains 3 potential N-linked glycosylation sites in thefirst extracellular region and 3 more in the third extracellular loop.

FIG. 3a depicts the flow cytometry determination of the intracellularCa⁺⁺ response of transfected human IL-8 and fMLP receptors to theirligands. Human embryonic kidney 293 cells were transfected byelectroporation (Gearing et al. EMBO J., 8: 3667-3676 1989!) with IL-8receptor (clone pRK5B.il8r1.1), fMLP receptor (human fMLP receptor cDNABoulay et al., Biochem. Biophys. Res. Comm., 168: 1103-1109 (1990)! inthe vector pRK5), or vector (pRK5B; EP 307,247) DNA. After two days, thecells were loaded with 2 μM indo-1 acetoxymethyl ester in RPMI medium(Sigma) for 30 min at 37° C. Intracellular Ca⁺⁺ was measured with aCoulter 753 flow cytometer using the ratio of 405 and 525 nmfluorescence. Grynkiewicz et al., J. Biol. Chem., 260: 3440-3450 (1985).

FIG. 3b illustrates the percent of cells above 400 nM Ca_(i) ⁺⁺ for thetime period after addition of IL-8 (about 15 sec. into each run).

FIGS. 4a-c (hereinafter collectively referred to as FIG. 4) depict theDNA sequence (SEQ ID NO.3) and an imputed polypeptide sequence (SEQ IDNO.4) for an additional chemokine superfamily receptor identified byprobing lambda libraries from a human monocyte-like cell line (HL-60)and human PBLs using a large fragment of the IL-8 receptor DNA.

FIGS. 5a-c (hereinafter collectively referred to as FIG. 5) depict theDNA sequence (SEQ ID NO. 5) and an imputed polypeptide sequence (SEQ IDNO.6) for yet another chemokine superfamily receptor identified byprobing lambda libraries from a human monocyte-like cell line (HL-60)and human PBLs using a large fragment of the IL-8 receptor DNA

FIG. 6 depicts the structure of the human IL-8 receptor A. 0:glycosylation site. Synthetic peptides covering the extracellular domainof the IL8R-A receptor were made to cover amino acids 2-19, 12-31,99-110, 176-187, 187-203, 265-277, and 277-291.

FIGS. 7A, 7B, 7C, and 7D disclose the binding of monoclonal antibody 2A4to transfected 293 cells expressing IL8R-A (293-71) (FIG. 7A),transfected 293 cells expressing IL8R-B (293-27) (FIG. 7B),untransfected 293 cells (FIG. 7C), and human neutrophils (FIG. 7D). Thesolid black line is the Mab 2A4 plus F-GamIg and the gray line is no MAbplus F-GamIg (control).

FIGS. 8A and 8B show the inhibition of ¹²⁵ I-labeled IL-8 binding tohuman neutrophils (FIG. 8A) and to 293 transfected cells expressingIL8R-A (293-17) (FIG. 8B) by various concentrations of 2A4 (filledcircles), 9H1 (open circles), IgG (open triangles), and, for FIG. 8B, noantibody (filled squares). The experiment using human neutrophils wascarried out in the presence of various concentrations of MGSA.

FIGS. 9A and 9B show the binding of monoclonal antibodies 2A4, 9H1, 4C8,6E9, and an IgG1 control to various synthetic peptides as determined byELISA. ELISA plates were coated with 2 μg/ml of peptides. Experimentswere done in triplicates. In FIG. 9A the solid bars are peptide 2-19,the diagonal hatched bars to the right of the solid bar are peptide12-31, the dark cross-hatching is peptide 99-100, the diagonal hatchingto the right of peptide 99-100 is peptide 176-187, the open bars arepeptide 187-203, the dotted bars are peptide 264-276, and the horizontalstriped bars are peptide 276-290. In FIG. 9B the solid bars are peptide2-19, the open bars are peptide 1-14, the dotted bars are peptide 1-11,and the diagonal hatching is peptide 1-13 (IL8R-B).

FIG. 10 shows the concentrations of IL-8 in sputum from various patientswith chronic airway inflammation (cystic fibrosis, bronchiectasis, andchronic bronchitis) and induced sputum for healthy subjects, where theopen squares are in-patients and the shaded squares are out-patients.

DETAILED DESCRIPTION OF THE INVENTION

By expression cloning was isolated a cDNA encoding the human neutrophilIL-8 receptor together with two other homologous receptors. The aminoacid sequence shows that the IL-8 receptor is a member of the G-proteincoupled receptor family with clear similarity (29% amino acid identity)to the human neutrophil receptors for the chemoattractants f-Met-Leu-Phe(Boulay et al., supra) and C5a (Gerard and Gerard, Nature, 349: 614-6171991!). Although the IL-8 receptor sequence may be the human homologueof what has been identified as the isoform of the rabbit f-Met-Leu-Phereceptor (Thomas et al., J. Biol. Chem., 265: 20061-20064 1990!), thisinvention shows that when transfected into mammalian cells, thisreceptor clone confers high affinity binding to IL-8 and produces atransient Ca⁺⁺ mobilization in response to IL-8 with no binding orresponse to f-Met-Leu-Phe.

A COS cell expression cloning strategy (Sims et al. , Science, 241:585-589 1988!); D'Andrea et al., Cell, 57: 277-285 1989!) was used toisolate clones encoding the IL-8 receptor. A cDNA library constructedfrom human neutrophil mRNA in the mammalian expression vector pRK5B wastransfected into COS-7 cells as pools of 2500 clones, and the cellsscreened for the binding of ¹²⁵ I-IL-8. One positive pool from the first58 transfections was partitioned into smaller pools until a pure clone(pRK5B.il8r1.1) was obtained. FIG. 1 shows the competition of ¹²⁵ I-IL-8binding by unlabelled IL-8 to COS cells transfected with the isolatedclone. Analysis of this data gives a Kd of 3.6 nM for IL-8 binding whichis within the range of 0.8 to 4 nM reported for IL-8 binding to humanneutrophils. Samanta et al., supra, Besemer et al., supra, Grob et al.,supra. There is no competition of the IL-8 binding by the chemotacticpeptide f-Met-Leu-Phe (fMLP).

The DNA sequence of the isolated cDNA clone (FIG. 2) contains a singlelong open reading frame beginning with a methionine residue that matchesthe consensus expected for a translation initiation site. Kozak, NucleicAcid Res., 12: 857-872 (1984). This open reading frame encodes a proteinof 350 amino acids (translated M_(r) 39.5 kD). The amino acid sequenceshares several features with the G-protein coupled receptors of therhodopsin superfamily including seven hydrophobic domains that arepresumed to span the cell membrane and N-linked glycosylation sites nearthe N-terminus (Dixon et al., Cold Spring Harb. Sym. Ouant. Biol., 53:487-497 1988!) (see below).

The encoded amino acid sequence is the most similar to a recently clonedsequence for the rabbit fMLP receptor. Thomas et al., supra. Thesimilarity is sufficiently high (79% amino acid identity overall withmultiple stretches of more than 20 contiguous amino acid matches) thatthese two sequences may well be species homologs of the same receptor.The human fMLP receptor has also been cloned (Boulay et al., supra); ithas only 26% amino acid identity with the rabbit fMLP receptor (and 29%identity to the human IL-8 receptor presented here). The considerabledivergence between the rabbit and human fMLP receptor amino acidsequences has lead to the suggestion in the art (now believed to bepossibly erroneous) that these may be two isoforms of the fMLP receptor.Thomas et al., supra.

Neutrophils respond to the chemoattractants IL-8 and fMLP with a rapid,transient increase in the intracellular free Ca⁺⁺ concentration.Oppenheim et al., supra; Korchak et al., J. Biol Chem., 259: 4076-4082(1984). In order to verify the identification of the clone isolated hereas the IL-8 receptor, we have determined the intracellular Ca⁺⁺ responseof transfected cells to added IL-8 as well as fMLP. We have usedparallel experiments with transfected human fMLP receptor or with theexpression vector as controls. Flow cytometer analysis shows a cleartransient increase in intracellular Ca⁺⁺ for the transfected IL-8receptor in response to IL-8. No response is found to fMLP. Conversely,cells transfected with the human fMLP receptor respond to fMLP but notto IL-8. No response to either chemoattractant is found in vectortransfected cells. Only a subset of the cells are expected to respond inthese experiments as the transfection efficiency is estimated to be15-25%. Binding experiments (Tennenberg et al., J. Immunol., 141:3937-3944 1988!) also failed to detect any binding of ³ H-fMLP to theexpressed IL-8 receptor or ¹²⁵ I-IL-8 to the expressed human fMLPreceptor. These experiments clearly demonstrate the specificity of thetwo receptors for their respective ligands; a result expected based onthe lack of binding competition between IL-8 and fMLP for neutrophils.Oppenheim et al., supra. These results also demonstrate that the clonedreceptors function in second message signaling in response to ligandbinding.

Blot hybridization of the cloned IL-8 receptor cDNA to human neutrophilmRNA, shows strong bands of 2.4 and 3.0 kb as well as a fainter band at3.5 kb. While it is clear from the DNA sequence data presented in FIG. 2that the mRNA for the receptor has a long 3' untranslated region,additional work will be needed to establish whether the multiple RNAbands are due to multiple polyadenylationsites. No hybridizationwasdetected to mRNA from U266 or Jurkat cell lines, which are of the B celland T cell lineages. No hybridization was found for mRNA from themonocyte cell line U937 as well, in spite of the reports of low levelsof IL-8 binding to these cells. Besemer et al., supra; Grob et al.,supra.

Alignment of the receptor sequences for the three neutrophilchemoattractants IL-8, fMLP (Boulay et al., supra), and C5a (Gerard andGerard, supra) shows that they form a subfamily of the G-protein coupledreceptors with 29-34% amino acid identity. This subfamily has a shortthird intracellular loop as compared with other G-protein coupledreceptors such as the β-adrenergic (Dixon et al., supra) or muscarinicacetylcholine receptors. Ramachandran, et al., BioEssays, 10: 54-57(1989). This loop contains determinants at least partially responsiblefor the binding of G-proteins to the receptors. Dixon et al., supra. Theintracellular C-terminal region of the IL-8 receptor, while not verysimilar to that of the fMLP and C5a receptors does preserve a highnumber of serine and threonine residues that may function asphosphorylation sites. As has been noted for the C5a receptor (Gerardand Gerard, supra), the N-terminal extracellular region for the IL-8receptor has several acidic residues. These may aid in the binding ofIL-8, which is quite basic (pI˜9.5).

I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims:

The term "PF4AR" is defined as a polypeptide having a qualitativebiological activity in common with the polypeptides of FIGS. 2, 4, or 5.Optionally, PF4AR will have at least 30% and ordinarily 75% amino acidsequence identity with any of the polypeptides of FIGS. 2, 4 or 5.Optionally, PF4AR excludes the rabbit fMLP receptor (Thomas et al.,supra), the human fMLP receptor (Boulay et al., supra), the human C5areceptor (Gerard and Gerard, supra), and/or the receptor described byMurphy and Tiffany, supra.

Identity or homology with respect to a PF4AR is defined herein to be thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in FIGS. 2, 4 or 5 after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions asrepresenting residue identity. No N- nor C-terminal extensions,deletions nor insertions shall be construed as reducing identity orhomology.

PF4AR qualitative biological activity is defined as any one of (1)immunological cross-reactivity with at least one epitope of apolypeptide set forth in FIGS. 2, 4, or 5; (2) the ability tospecifically bind to a member of the PF4 superfamily; or (3) anyeffector or functional activity of the FIGS. 2, 4 or 5 polypeptides asfound in nature, including their ability to bind any ligands other thansuperfamily members.

Immunologically cross-reactive as used herein means that the candidatepolypeptide is capable of competitively inhibiting the binding of aPF4AR to polyclonal antibodies or antisera raised against a PF4AR. Suchantibodies and antisera are prepared in conventional fashion byinjecting an animal such as a goat or rabbit, for example,subcutaneously with the known native PF4AR in complete Freund'sadjuvant, followed by booster intraperitoneal or subcutaneous injectionin incomplete Freund's.

Included within the scope of the PF4AR as that term is used herein arepolypeptides having the amino acid sequences described in FIGS. 2, 4 or5, amino acid sequence variants of such amino acid sequences,glycosylation variants of the polypeptides and covalent modifications ofthe polypeptides. Each of these are described in more detail below.

"Isolated" PF4AR nucleic acid or polypeptide is a PF4AR nucleic acid orpolypeptide that is identified and separated from at least onecontaminant (nucleic acid or polypeptide respectively) with which it isordinarily associated in nature, such as from the animal or human sourceof the PF4AR nucleic acid or polypeptide. In preferred embodiments, thePF4AR will be isolated to pharmaceutically acceptable levels of puritywith respect to proteins of its species of origin. In preferredembodiments, PF4AR protein will be purified (1) to greater than 95% byweight of protein as determined by the Lowry method, and most preferablymore than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by an aminoacid sequenator commercially available on the filing date hereof, or (3)to homogeneity by conventional nonreducing SDS-PAGE using Coomassie blueor, preferably, silver stain. Isolated PF4AR includes PF4AR in situwithin recombinant cells since, in this instance, at least one componentof the PF4AR natural environment will not be present. Isolated PF4ARincludes PF4AR from one species in a recombinant cell culture of anotherspecies since the receptor in such circumstances will be devoid ofsource polypeptides. Ordinarily, however, isolated receptor will beprepared by at least one purification step.

Isolated PF4AR nucleic acid includes a nucleic acid that is identifiedand separated from at least one containment nucleic acid with which itis ordinarily associated in the natural source of the receptor nucleicacid. Isolated PF4AR nucleic acid thus is present in other than in theform or setting in which it is found in nature. However, isolatedreceptor-encoding nucleic acid includes PF4AR nucleic acid in ordinarilyreceptor-expressing cells where the nucleic acid is in a chromosomallocation different from that of natural cells or is otherwise flanked bya different DNA sequence than that found in nature.

The nucleic acid or polypeptide may be labeled for diagnostic and probepurposes, using a label as described and defined further below in thediscussion of diagnostic assays.

PF4AR "nucleic acid" is defined as RNA or DNA containing greater thanten bases that encodes a polypeptide sequence within FIGS. 2, 4 or 5, iscomplementary to nucleic acid sequence of FIGS. 2, 4 or 5, hybridizes tosuch nucleic acid and remains stably bound to it under low stringencyconditions, or encodes a polypeptide sharing at least 30% sequenceidentity, preferably at least 75%, and more preferably at least 85%,with the translated amino acid sequence shown in FIGS. 2, 4 or 5 or afragment thereof. Preferably the DNA which hybridizes to the nucleicacid of FIGS. 2, 4 or 5 contain at least 20, more preferably 40, andmore preferably 60 bases. Most preferably, the hybridizing DNA or RNAcontains or even more preferably 90 bases. Such hybridizing orcomplementary nucleic acid, however, is defined further as being noveland unobvious over any prior art nucleic acid including that whichencodes, hybridizes under low stringency conditions, or is complementaryto nucleic acid encoding rabbit fMLP receptor (Thomas et al., supra),human fMLP receptor or (optionally) the IL-8 receptor of Murphy andTiffany, supra.

"High stringency conditions" are any of those that (1) employ low ionicstrength and high temperature for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ at 50° C.; (2) employ duringhybridization 50% (vol/vol) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or(3) employ hybridization with 50% formamide, 5×SSC (0.75 M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1 SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS. Conditions of low stringency are set forthin Example 2.

The term "control sequences" refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Howeverenhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

The starting plasmids herein are commercially available, are publiclyavailable on an unrestricted basis, or can be constructed from suchavailable plasmids in accord with published procedures. In addition,other equivalent plasmids are known in the art and will be apparent tothe ordinary artisan. Methods for restriction enzyme digestion, recoveryor isolation of DNA, hybridization analysis, and ligation areconventional and by this time well known to the ordinary artisan.

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114 (1981), and Goeddel et al., Nucleic Acids Res., 8: 4057(1980).

As used herein, the term "mammal" refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal herein is human.

As used herein, the term "treatment" refers to therapy as well asprophylactic (preventative) measures.

As used herein, the term "inflammatory disorders" refers to pathologicalstates resulting in inflammation, typically caused by neutrophilchemotaxis. Examples of such disorders include T cell inflammatoryresponses such as inflammatory skin diseases including psoriasis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); adult respiratory distress syndrome;dermatitis; meningitis; encephalitis; uveitis; autoimmune diseases suchas rheumatoid arthritis and Sjorgen's syndrome, diseases involvingleukocyte diapedesis; CNS inflammatory disorder, multiple ischemiareperfusion injury, traumatic shock, hypovolemic shock, organ injurysyndrome secondary to septicaemia or trauma; alcoholic hepatitis;antigen-antibody complex mediated diseases; inflammations of the lung,including pleurisy, alveolitis, vasculitis, pneumonia, chronicbronchitis, bronchiectasis, and cystic fibrosis; etc. The preferredindications are inflammatory bowel disease such as ulcerative colitis ora chronic lung inflammation.

II. Suitable Methods for Practicing the Invention

1. Preparation of Native PF4AR and Variants

A. Isolation of DNA Encoding PF4AR

The DNA encoding of the PF4AR may be obtained from any cDNA libraryprepared from tissue believed to contain the PF4AR mRNA, generally HL60or PBL libraries. The PF4AR gene may also be obtained from a genomiclibrary. Libraries are screened with probes designed to identify thegene of interest or the protein encoded by it. The entire cDNA for theIL-8 receptor and two homologous receptors is described. Nucleic acidencoding this family of receptors is readily obtained under lowstringency conditions from genomic DNA or neutrophil cDNA librariesusing probes having oligonucleotide sequences from the receptor genesequences of FIGS. 2, 4 or 5. These probes usually will contain about500 or more bases. Since the probes will hybridize perfectly to thethree exemplified DNAs, there is no need to use probe pools containingdegenerate sequences. Screening with the probes to identify the FIGS. 2,4 or 5 receptors is more efficient if performed under conditions of highstringency.

Other PF4ARs other than those in FIGS. 2, 4 or 5 are believed to existand to contain regions of homology to the exemplified receptors. Thusprobes having the sequences of the DNAs in FIGS. 2, 4 or 5 can be usedto screen for these receptors as well. The best candidates for probesare long sequences (greater than about 100 bases) that representsequences that are highly homologous among the three exemplified humanreceptors. IL-8 receptor cDNA encoding the IL-8 receptor residues 15-34,78-94, 176-193, 264-282 and 299-312 (and comparable probes from otherreceptors of the IL-8R family) are useful, particularly in probing forIL-8 receptor DNA. Probes useful for the receptor of FIG. 4 (andisolated proteins characteristic of the FIG. 5 receptor) are representedby sequences comprising residues 1-48, 77-92, 107-137, 156-177, 189-226,239-257 and 271-315. Homologous probes and residues of the FIG. 4receptor also are useful, i.e. residues 1-35, 64-78, 94-124, 143-164,176-197, 219-239 and 251-295. cDNAs comprising cDNA encoding thefollowing regions of the FIGS. 2, 4 or 5 polypeptides are useful inprobing for other receptors: 92-106, 57-72, 138-154, 314-329 and 57-154.

In general, one first identifies a cell which is capable of specificallybinding or which is activated by a given PF4A, typically by in vitrobioassays and, optionally, by cell binding analysis using the labelledPF4A. Cells identified by this process (and some are already known forindividual PF4As) therefore are expressing a receptor for this PF4A. AcDNA library is prepared from such cells and is screened using thereceptor probes by procedures that are conventional per se. In thisinstance, however, it is preferred to use low stringency conditions(such as those in Example 2) and then analyze the resulting positiveclones for homology to the FIGS. 2, 4 or 5 receptors. In general,candidate human PF4ARs will exhibit greater than about 30% amino acidsequence homology to the FIGS. 2, 4 or 5 receptors and bear a similartransmembrane loop structure.

Assays are then conducted to confirm that the hybridizing full lengthgenes are the desired PF4AR. The candidate is simply inserted into anexpression vector and transformed into a host cell that ordinarily doesnot bind to the candidate PF4A ligand. Transformants that acquire theability to bind the ligand thus bear the desired receptor gene. InExample 2, we show that two additional homologous polypeptide sequencesrepresenting PR4ARs are identified using IL-8R DNA encoding residues23-314, although the particular probe is not believed to be critical.

An alternative means to isolate genes encoding additional PF4ARs is touse polymerase chain reaction (PCR) methodology (U.S. Pat. No.4,683,195; Erlich, ed., PCR Technology, 1989) to amplify the target DNAor RNA, e.g. as described in section 14 of Sambrook et al., supra. Thismethod requires the use of oligonucleotide primers that will be expectedto hybridize to the PF4AR, and these readily are selected from thereceptor cDNAs of FIGS. 2, 4 or 5. Strategies for selection ofoligonucleotide primers are described above.

cDNA libraries may be screened from various tissues, preferablymammalian PBL, monocyte, placental, fetal, brain, and carcinoma celllines in order to obtain DNA encoding the receptors of FIGS. 2, 4 or 5,or homologous receptors. More preferably, human or rabbit placental,fetal, brain, and carcinoma cell line cDNA libraries are screened withlabelled oligonucleotideprobes. Another method for obtaining the gene ofinterest is to chemically synthesize it using one of the methodsdescribed in Engels et al., Agnew. Chem. Int. Ed. Enql., 28: 716-734(1989). These methods include triester, phosphite, phosphoramidite andH-phosphonate methods, typically proceeding by oligonucleotide synthesison solid supports. These methods may be used if the entire amino acid ornucleic acid sequence of the gene is known, or the sequence of thenucleic acid complementary to the coding strand is available. If thedesired amino acid sequence is known, one may infer potential nucleicacid sequences using known and preferred coding residues for each aminoacid residue.

B. Amino Acid Sequence Variants of the PF4AR

Amino acid sequence variants of the PF4AR are prepared by introducingappropriate nucleotide changes into the PF4AR DNA, or by in vitrosynthesis of the desired PF4AR polypeptide. Such variants include, forexample, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence shown for the receptors in FIGS. 2, 4 or5. Any combination of deletion, insertion, and substitution can be madeto arrive at the final construct, provided that the final constructpossesses the desired characteristics.

The amino acid changes also may alter post-translational processing ofthe PF4AR, such as changing the number or position of glycosylationsites or by altering its membrane anchoring characteristics. Excludedfrom the scope of this invention are PF4AR variants or polypeptidesequences that are not statutorily novel and unobvious over the priorart.

In designing amino acid sequence variants of PF4ARs, the location of themutation site and the nature of the mutation will depend on the PF4ARcharacteristic(s) to be modified. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconservative amino acid choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue, or(3) inserting residues of the same or a different class adjacent to thelocated site, or combinations of options 1-3.

A useful method for identification of certain residues or regions of thePF4AR polypeptide that are preferred locations for mutagenesis is called"alanine scanning mutagenesis" as described by Cunningham and Wells,Science, 244: 1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the surrounding aqueous environment in or outsidethe cell. Those domains demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, ala scanning orrandom mutagenesis may be conducted at the target codon or region andthe expressed PF4AR variants are screened for the optimal combination ofdesired activity.

In general, the regions of the PF4AR molecule preferred for alterationsare non-hydrophilic regions or regions that are not highly conserved.Such regions are those in which sequences of 5 or more residues are notsubstantially conserved in the homologous positions in the rabbit fMLPreceptor, the human fMLP receptor, the human C5a receptor and thereceptors of FIGS. 2, 4 and 5.

PF4AR variants will exhibit at least a biological activity of theparental sequence, for example ligand binding activity or antigenicactivity. Antigenically active PF4AR is a polypeptide that binds with anaffinity of at least about 10⁻⁹ l/mole to an antibody raised against anaturally occurring PF4AR sequence. Ordinarily the polypeptide bindswith an affinity of at least about 10⁻⁸ l/mole. Most preferably, theantigenically active PF4AR is a polypeptide that binds to an antibodyraised against the receptor in its native conformation, "nativeconformation" generally meaning the receptor as found in nature whichhas not been denatured by chaotropic agents, heat or other treatmentthat substantially modifies the three dimensional structure of thereceptor (this can be determined, for example, by migration onnonreducing, nondenaturing sizing gels). Antibody used in determinationof antigenic activity is rabbit polyclonal antibody raised byformulating the native non-rabbit receptor in Freund's completeadjuvant, subcutaneously injecting the formulation, and boosting theimmune response by intraperitoneal injection of the formulation untilthe titer of antireceptor antibody plateaus.

One group of variants are deletion mutants, or fragments of thesequences set forth in FIGS. 2, 4, 5 or other PF4AR. In general, thefragments are those which constitute the extracellular regions of thereceptors (these receptors are unlike most in that they are believed tocontain a plurality of hydrophobic, trans-membrane domains separated byhydrophilic sequences believed to loop into the ectoplasm). Particularlyof interest are the N-terminal extracellular region containing acidicamino acid residues. However, any sequence which is capable of raisingan antibody that will cross-react with the intact receptor, or whichwill bind to a member of the PF4 superfamily, is useful. These fragmentstypically will contain a consecutive sequence of at least about 5 (andordinarily at least about 10) residues.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions of low homologyamong the receptors of FIGS. 2, 4 and 5 to modify the activity of thereceptors. Such deletions will be more likely to modify the biologicalactivity of the receptors more significantly than deletions madeelsewhere. The number of consecutive deletions will be selected so as topreserve the tertiary structure of the PF4AR in the affected domain,e.g., beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the PF4AR sequence) may range generally from about 1to 10 residues, more preferably 1 to 5, most preferably 1 to 3.

Insertional variants of the PF4AR or its extracellular segments includethe fusion to the N- or C-terminus of the PF4AR of immunogenicpolypeptides, e.g., bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin constant regions (or other immunoglobulin regions),albumin, or ferritin, as described in WO 89/02922 published Apr 6, 1989.

Another group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the PF4AR moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of the PF4AR, and sites where the aminoacids found in the PF4AR from various species are substantiallydifferent in terms of side-chain bulk, charge, and/or hydrophobicity.

Other sites of interest are those in which particular residues of thePF4ARs of FIGS. 2, 4 and 5 are identical. These positions may beimportant for the biological activity of the PF4AR. These sites,especially those falling within a sequence of at least three otheridentically conserved sites, are substituted in a relativelyconservative manner. Such conservative substitutions are shown in Table1 under the heading of preferred substitutions. If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated exemplary substitutions in Table 1, or as furtherdescribed below in reference to amino acid classes, are introduced andthe products screened.

                  TABLE 1    ______________________________________    Original    Exemplary     Preferred    Residue     Substitutions Substitutions    ______________________________________    Ala (A)     val; leu; ile val    Arg (R)     lys; gln; asn lys    Asn (N)     gln; his; lys; arg                              gln    Asp (D)     glu           glu    Cys (C)     ser           ser    Gln (Q)     asn           asn    Glu (E)     asp           asp    Gly (G)     pro           pro    His (H)     asn; gln; lys; arg                              arg    Ile (I)     leu; val; met; ala; phe;                norleucine    leu    Leu (L)     norleucine; ile; val;                met; ala; phe ile    Lys (K)     arg; gln; asn arg    Met (M)     leu.; phe; ile                              leu    Phe (F)     leu; val; ile; ala                              leu    Pro (P)     gly           gly    Ser (S)     thr           thr    Thr (T)     ser           ser    Trp (W)     tyr           tyr    Tyr (Y)     trp; phe; thr; ser                              phe    Val (V)     ile; leu; met; phe;                ala; norleucine                              leu    ______________________________________

Substantial modifications in function or immunological identity of thePF4AR are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another. Such substituted residues may be introducedinto regions of the PF4AR that are homologous with other PF4ARs, or,more preferably, into the non-homologous regions of the molecule.

Any cysteine residues not involved in maintaining the properconformation of the PF4AR may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking.

DNA encoding amino acid sequence variants of the PF4AR is prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the PF4AR. These techniques may utilize PF4ARnucleic acid (DNA or RNA), or nucleic acid complementaryto the PF4ARnucleic acid.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion variants of PF4AR DNA. Thistechnique is well known in the art, for example as described by Adelmanet al., DNA, 2: 183 (1983). Briefly, the PF4AR DNA is altered byhybridizing an oligonucleotide encoding the desired mutation to a DNAtemplate, where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thePF4AR. After hybridization, a DNA polymerase is used to synthesize anentire second complementary strand of the template that will thusincorporate the oligonucleotide primer, and will code for the selectedalteration in the PF4AR DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. USA, 75: 5765 (1978).

Single-stranded DNA template may also be generated by denaturingdouble-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the PF4AR, and the other strand (the original template) encodesthe native, unaltered sequence of the PF4AR. This heteroduplex moleculeis then transformed into a suitable host cell, usually a prokaryote suchas E. coli JM101. The cells are plated onto agarose plates, and screenedusing the oligonucleotide primer radiolabeled with 32-phosphate toidentify the bacterial colonies that contain the mutated DNA. Themutated region is then removed and placed in an appropriate vector forprotein production, generally an expression vector of the type typicallyemployed for transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham Corporation). This mixture is added to thetemplate-oligonucleotidecomplex. Upon addition of DNA polymerase to thismixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(as) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion. After the template strand of thedouble-stranded heteroduplex is nicked with an appropriate restrictionenzyme, the template strand can be digested with ExoIII nuclease oranother appropriate nuclease past the region that contains the site(s)to be mutagenized. The reaction is then stopped to leave a molecule thatis only partially single-stranded. A complete double-stranded DNAhomoduplex is then formed using DNA polymerase in the presence of allfour deoxyribonucleotide triphosphates, ATP, and DNA ligase. Thishomoduplex molecule can then be transformed into a suitable host cellsuch as E. coli JM101, as described above.

DNA encoding PF4AR mutants at more than one site may be generated in oneof several ways. If the amino acids are located close together in thepolypeptide chain, they may be mutated simultaneously using oneoligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom each other (separated by more than about ten amino acids), it ismore difficult to generate a single oligonucleotide that encodes all ofthe desired changes. Instead, one of two alternative methods may beemployed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions. The alternative method involves two ormore rounds of mutagenesis to produce the desired mutant. The firstround is as described for the single mutants: wild-type DNA is used forthe template, an oligonucleotide encoding the first desired amino acidsubstitution(s) is annealed to this template, and the heteroduplex DNAmolecule is then generated. The second round of mutagenesis utilizes themutated DNA produced in the first round of mutagenesis as the template.Thus, this template already contains one or more mutations. Theoligonucleotide encoding the additional desired amino acidsubstitution(s) is then annealed to this template, and the resultingstrand of DNA now encodes mutations from both the first and secondrounds of mutagenesis. This resultant DNA can be used as a template in athird round of mutagenesis, and so on.

PCR mutagenesis is also suitable for making amino acid variants of thePF4AR. While the following discussion refers to DNA, it is understoodthat the technique also finds application with RNA. The PCR techniquegenerally refers to the following procedure (see Erlich, supra, thechapter by R. Higuchi, p. 61-70): When small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template. For introduction of a mutation into aplasmid DNA, one of the primers is designed to overlap the position ofthe mutation and to contain the mutation; the sequence of the otherprimer must be identical to a stretch of sequence of the opposite strandof the plasmid, but this sequence can be located anywhere along theplasmid DNA. It is preferred, however, that the sequence of the secondprimer is located within 200 nucleotides from that of the first, suchthat in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the position of the mutation specified by the primer, andpossibly at other positions, as template copying is somewhaterror-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene, 34: 315 (1985).

C. Insertion of DNA into a Cloning Vehicle

The cDNA or genomic DNA encoding native or variant PF4AR is insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. Many vectors are available, and selection of theappropriate vector will depend on (1) whether it is to be used for DNAamplification or for DNA expression, (2) the size of the DNA to beinserted into the vector, and (3) the host cell to be transformed withthe vector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the host cellfor which it is compatible. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(i) Signal Sequence Component

In general, a signal sequence may be a component of the vector, or itmay be a part of the PF4AR DNA that is inserted into the vector. Thenative pro PF4AR DNA is directed to the cell surface in our recombinantcells but it does not contain a conventional signal and no N-terminalpolypeptide is cleaved during post-translational processing of thepolypeptide during membrane insertion of the PF4AR.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are "shuttle" vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the PF4AR DNA. However, the recovery of genomic DNAencoding the PF4AR is more complex than that of an exogenouslyreplicated vector because restriction enzyme digestion is required toexcise the PF4AR DNA.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, ortetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g. the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 3271982!), mycophenolic acid (Mulligan et al., Science, 209: 1422 1980!) orhygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 19851). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePF4AR nucleic acid, such as dihydrofolate reductase (DHFR) or thymidinekinase. The mammalian cell transformants are placed under selectionpressure which only the transformants are uniquely adapted to survive byvirtue of having taken up the marker. Selection pressure is imposed byculturing the transformants under conditions in which the concentrationof selection agent in the medium is successively changed, therebyleading to amplification of both the selection gene and the DNA thatencodes the PF4AR. Amplification is the process by which genes ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of the PF4AR are synthesizedfrom the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 (1980). The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingthe PF4AR. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-Kl, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060). Alternatively, host cells(particularly wild-type hosts that contain endogenous DHFR) transformedor co-transformed with DNA sequences encoding the PF4AR, wild-type DHFRprotein, and another selectable marker such as aminoglycoside 3'phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7. Stinchcomb et al., Nature, 282: 39 (1979);Kingsman et al., Gene, 7: 141 (1979); or Tschemper et al., Gene, 10: 157(1980). The trp1 gene provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression vectors usually contain a promoter that is recognized by thehost organism and is operably linked to the PF4AR nucleic acid.Promoters are untranslated sequences located upstream (5') to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of a particular nucleic acidsequence, such as the PF4AR, to which they are operably linked. Suchpromoters typically fall into two classes, inducible and constitutive.Inducible promoters are promoters that initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g. the presence or absence of a nutrient or achange in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known. Thesepromoters are operably linked to DNA encoding the PF4AR by removing thepromoter from the source DNA by restriction enzyme digestion andinserting the isolated promoter sequence into the vector. Both thenative PF4AR promoter sequence and many heterologous promoters may beused to direct amplification and/or expression of the PF4AR DNA.However, heterologous promoters are preferred, as they generally permitgreater transcription and higher yields of expressed PF4AR as comparedto the native PF4AR promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275: 6151978!; and Goeddel et al., Nature, 281: 544 1979!), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 1980! and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983!).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the PF4AR (Siebenlist et al.,Cell, 20: 269 1980!) using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also generallywill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the PF4AR.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem.,255: 2073 1980!) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Req., 7: 149 1968!; and Holland, Biochemistry, 17: 4900 1978!),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mmutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also areadvantageouslyused with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

PF4AR transcription from vectors in mammalian host cells is controlledby promoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus (UK 2,211,504 published Jul. 5, 1989, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g. theactin promoter or an immunoglobulin promoter, from heat-shock promoters,and from the promoter normally associated with the PF4AR sequence,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273: 113 (1978); Mulligan andBerg, Science, 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad.Sci. USA, 78: 7398-7402 (1981). The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment. Greenaway et al., Gene, 18: 355-360 (1982). Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso Gray et al., Nature, 295: 503-508 (1982) on expressing cDNAencoding immune interferon in monkey cells, Reyes et al., Nature, 297:598-601 (1982) on expression of human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus, Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170(1982) on expression of the human interferon β1 gene in cultured mouseand rabbit cells, and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the PF4AR of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from10-300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent havingbeen found 5' (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 9931981!) and 3' (Lusky et al., Mol. Cell Bio., 3: 1108 1983!) to thetranscription unit, within an intron (Banerji et al., Cell, 33: 7291983!) as well as within the coding sequence itself (Osborne et al.,Mol. Cell Bio., 4: 1293 1984!). Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' to thePF4AR DNA, but is preferably located at a site 5' from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3' untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the PF4AR. The 3' untranslated regions alsoinclude transcription termination sites.

Suitable vectors containing one or more of the above listed componentsand the desired coding and control sequences are constructed by standardligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the PF4AR. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of the PF4AR that have PF4AR-likeactivity.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the PF4AR in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293: 620-625 (1981); Mantei et al.,Nature, 281: 40-46 (1979); Levinson et al., EP 117,060; and EP 117,058.A particularly useful plasmid for mammalian cell culture expression ofthe PF4AR is pRK5 (EP pub. no. 307,247) or pSVI6B (PCT pub. no. WO91/08291 published Jun. 13, 1991).

D. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescens. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli X1776(ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting. Preferably the host cellshould secrete minimal amounts of proteolytic enzymes. Alternatively, invitro methods of cloning, e.g. PCR or other nucleic acid polymerasereactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors containing PF4AR DNA.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as S. pombe (Beach and Nurse, Nature, 290: 140 1981!),Kluyveromyces lactis (Louvencourt et al., J. Bacteriol., 737 1983!),yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Trichoderma reesia(EP 244,234), Neurospora crassa (Case et al., Proc. Natl. Acad. Sci.USA, 76: 5259-5263 1979!), and Aspergillus hosts such as A. nidulans(Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 1983!;Tilburn et al., Gene, 26: 205-221 1983!; Yelton et al., Proc. Natl.Acad. Sci. USA, 81: 1470-1474 1984!) and A. niger (Kelly and Hynes, EMBOJ., 4: 475-479 1985!).

Suitable host cells for the expression of glycosylated PF4AR polypeptideare derived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori hostcells have been identified. See, e.g., Luckow et al., Bio/Technology, 6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow, J. K. etal., 8: 277-279 (Plenum Publishing, 1986), and Maeda et al., Nature,315: 592-594 (1985). A variety of such viral strains are publiclyavailable, e.g., the L-1 variant of Autographa californica NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the PF4AR DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding PF4AR is transferred to the plant cellhost such that it is transfected, and will, under appropriateconditions, express the PF4AR DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylationsignal sequences. Depickeret al., J. Mol. Appl. Gen., 1: 561 (1982). In addition, DNA segmentsisolated from the upstream region of the T-DNA 780 gene are capable ofactivating or increasing transcription levels of plant-expressible genesin recombinant DNA-containingplant tissue. See EP 321,196 published Jun.21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)!. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36: 59 (1977); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980!); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980!); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383: 44-68 1982!); MRC 5 cells; FS4 cells; and a human hepatomacell line (Hep G2). Preferred host cells are human embryonic kidney 293and Chinese hamster ovary cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 publishedJun. 29, 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method described in sections 16.30-16.37 ofSambrook et al., supra, is preferred. General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued August 16, 1983. Transformations into yeast aretypically carried out according to the method of Van Solingen et al., J.Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA),76: 3829 (1979). However, other methods for introducing DNA into cellssuch as by nuclear injection, electroporation, or by protoplast fusionmay also be used.

E. Culturing the Host Cells

Prokaryotic cells used to produce the PF4AR polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the PF4AR of this invention maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ( MEM!, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ( DMEM!, Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, Meth. Enz., 58: 44 (1979), Barnes andSato, Anal. Biochem., 102: 255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat.No. Re. 30,985; or U.S. Pat. No. 5,122,469, may be used as culture mediafor the host cells. Any of these media may be supplemented as necessarywith hormones and/or other growth factors (such as insulin, transferrin,or epidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

It is further envisioned that the PF4AR of this invention may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding the PF4AR. For example, a powerfulpromoter/enhancer element, a suppressor, or an exogenous transcriptionmodulatory element is inserted in the genome of the intended host cellin proximity and orientation sufficient to influence the transcriptionof DNA encoding the desired PF4AR. The control element does not encodethe PF4AR of this invention, but the DNA is present in the host cellgenome. One next screens for cells making the PF4AR of this invention,or increased or decreased levels of expression, as desired.

F. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 1980!), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³² P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native PF4AR polypeptide or against a synthetic peptide basedon the DNA sequences provided herein as described further in Section 4below.

G. Purification of The PF4AR Polypeptide

The PF4AR is recovered from the culture cells by solubilizing cellmembrane in detergent.

When a human PF4AR is expressed in a recombinant cell other than one ofhuman origin, the PF4AR is completely free of proteins or polypeptidesof human origin. However, it is necessary to purify the PF4AR fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to the PF4AR. As a first step, thecells are centrifuged to separate them from culture medium. The membraneand soluble protein fractions are then separated. The PF4AR may then bepurified from the membrane fraction of the culture lysate bysolubilization with detergents followed by suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; hydrophobic affinity resins and ligand affinity using theappropriate PF4A immobilized on a matrix.

PF4AR variants in which residues have been deleted, inserted orsubstituted are recovered in the same fashion as the native PF4AR,taking account of any substantial changes in properties occasioned bythe variation. For example, preparation of a PF4AR fusion with anotherprotein or polypeptide, e.g. a bacterial or viral antigen, facilitatespurification; an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion. Immunoaffinity columns such asa rabbit polyclonal anti-PF4AR column can be employed to absorb thePF4AR variant by binding it to at least one remaining immune epitope. Aprotease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native PF4AR may require modification to accountfor changes in the character of the PF4AR or its variants uponexpression in recombinant cell culture.

H. Covalent Modifications of PF4AR Polypeptides

Covalent modifications of PF4AR polypeptides are included within thescope of this invention. Both native PF4ARs and amino acid sequencevariants of the PF4AR may be covalently modified. Covalent modificationsof the PF4AR, fragments thereof or antibodies thereto are introducedinto the molecule by reacting targeted amino acid residues of the PF4AR,fragments thereof, or PF4AR antibody with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues. Most commonly, PF4AR and its antibodies arecovalently bonded to detectable groups used in diagnosis, e.g. enzymes,radio isotopes, spin labels, antigens, fluorescent or chemiluminescentgroups and the like.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidazole)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzedreactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R'), where R and R' aredifferent alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingPF4AR, its fragments or antibodies to a water-insoluble support matrixor surface for use in methods for purifying anti-PF4AR antibodies, andvice versa. Immobilized PF4AR also is useful in screening for the PF4superfamily members to which the receptor binds. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate),andbifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizingagents such as methyl-3- (p-azidophenyl)dithio!propioimidate yieldphotoactivatable intermediates that are capable of forming crosslinks inthe presence of light. Alternatively, reactive water-insoluble matricessuch as cyanogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440 are employed for proteinimmobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 1983!),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the beta-8 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. By altering is meant deletingone or more carbohydrate moieties found in the native receptor, and/oradding one or more glycosylation sites that are not present in thenative receptor.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. As noted above, the IL-8 receptorcontains 6 putative N-linked glycosylation sites.

Addition of glycosylation sites to the PF4AR polypeptide is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thenative PF4AR sequence (for O-linked glycosylation sites). For ease, thePF4AR amino acid sequence is preferably altered through changes at theDNA level, particularly by mutating the DNA encoding the PF4ARpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids. The DNA mutation(s) may bemade using methods described above under the heading of "Amino AcidSequence Variants of PF4AR Polypeptide".

Another means of increasing the number of carbohydrate moieties on thePF4AR polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. These procedures are advantageous in that they do notrequire production of the polypeptide in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (CRC Crit. Rev. Biochem., pp. 259-3061981!).

Removal of carbohydrate moieties present on the native PF4AR polypeptidemay be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonicacid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine) ,while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al.,Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydratemoieties on polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al., Meth.Enzymol., 138: 350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duskin et al., J. Biol.Chem., 257: 3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

The PF4AR also may be entrapped in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization (forexample, hydroxymethylcellulose or gelatin-microcapsules and poly-methylmethacylate! microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A., Ed., (1980).

PF4AR preparations are also useful in generating antibodies, for use asstandards in assays for the PF4AR (e.g. by labeling the PF4AR for use asa standard in a radioimmunoassay, enzyme-linked immunoassay, orradioreceptor assay), in affinity purification techniques, and incompetitive-type receptor binding assays when labeled with radioiodine,enzymes, fluorophores, spin labels, and the like.

Since it is often difficult to predict in advance the characteristics ofa variant PF4AR, it will be appreciated that some screening of therecovered variant will be needed to select the optimal variant. Forexample, a change in the immunological character of the PF4AR molecule,such as affinity for a given antibody, is measured by a competitive-typeimmunoassay. The variant is assayed for changes in the suppression orenhancement of its activity by comparison to the activity observed fornative PF4AR in the same assay. Other potential modifications of proteinor polypeptide properties such as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known in the art.

3. Therapeutic Compositions and Administration of PF4AR

Therapeutic formulations of PF4AR (including its PF4AR bindingfragments) or antibodies thereto are prepared for storage by mixingPF4AR having the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, supra), in the form of lyophilized cake oraqueous solutions. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

The PF4AR, or antibody to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The PF4AR ordinarily will be stored in lyophilized formor in solution.

Therapeutic PF4AR or antibody compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The route of PF4AR or antibody administration is in accord with knownmethods, e.g. injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, or intralesional routes, or by sustained releasesystems as noted below. Preferably the antibody is given systemically.

Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556 1983!), poly (2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 1981! and Langer,Chem. Tech., 12: 98-105 1982!), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).Sustained-release PF4AR or antibody compositions also includeliposomally entrapped PF4AR or antibody. Liposomes containing PF4AR orantibody are prepared by methods known per se: DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese patent application83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.ordinarily the liposomes are of the small (about 200-800 Angstroms)unilamelar type in which the lipid content is greater than about 30 mol.% cholesterol, the selected proportion being adjusted for the optimalPF4AR or antibody therapy.

PF4AR or antibody can also be administered by inhalation. Commerciallyavailable nebulizers for liquid formulations, including jet nebulizersand ultrasonic nebulizers are useful for administration. Liquidformulations can be directly nebulized and lyophilized powder can benebulized after reconstitution. Alternatively, PF4AR or antibody can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

An "effective amount" of PF4AR or antibody to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, the type of PF4AR polypeptideor antibody employed, and the condition of the patient. Accordingly, itwill be necessary for the therapist to titer the dosage and modify theroute of administration as required to obtain the optimal therapeuticeffect. Typically, the clinician will administer the PF4AR or antibodyuntil a dosage is reached that achieves the desired effect. The progressof this therapy is easily monitored by conventional assays.

In the treatment and prevention of an inflammatory disorder by a PF4ARantibody, particularly an IL-8 receptor antibody, the antibodycomposition will be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the antibody, theparticular type of antibody, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The "therapeutically effective amount" of antibody to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat the inflammatorydisorder, including treating chronic respiratory diseases and reducinginflammatory responses. Such amount is preferably below the amount thatis toxic to the host or renders the host significantly more susceptibleto infections.

As a general proposition, the initial pharmaceutically effective amountof the antibody administered parenterally per dose will be in the rangeof about 0.1 to 50 mg/kg of patient body weight per day, with thetypical initial range of antibody used being 0.3 to 20 mg/kg/day, morepreferably 0.3 to 15 mg/kg/day.

As noted above, however, these suggested amounts of antibody are subjectto a great deal of therapeutic discretion. The key factor in selectingan appropriate dose and scheduling is the result obtained, as indicatedabove.

The antibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the inflammatory disorder inquestion. For example, in rheumatoid arthritis, the antibody may begiven in conjunction with a glucocorticosteroid. In addition, T cellreceptor peptide therapy is suitably an adjunct therapy to preventclinical signs of autoimmune encephalomyelitis. Offner et al., supra.The effective amount of such other agents depends on the amount of PF4ARantibody present in the formulation, the type of disorder or treatment,and other factors discussed above. These are generally used in the samedosages and with administration routes as used hereinbefore or aboutfrom 1 to 99% of the heretofore employed dosages.

4. PF4AR Antibody Preparation

Polyclonal antibodies to the PF4AR generally are raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of thePF4AR and an adjuvant. Immunization with recombinant cells transformedwith the PF4AR (e.g., 293, mouse, or CHO cells transformed with huPF4AR)may be satisfactory, or it may be useful to separate the PF4AR andconjugate it or a fragment containing the target amino acid sequence toa protein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR,where R and R¹ are different alkyl groups.

Animals ordinarily are immunized against the transfected cells, orimmunogenic conjugates, derivatives, or peptides every other week. 7 to14 days after the immunization the animals are bled and the serum isassayed for anti-PF4AR titer. Preferably, the animal is boosted with theconjugate of the same PF4AR, but conjugated to a different proteinand/or through a different cross-linking agent. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are used to enhance the immune response. Lesspreferably, the animals are immunized by combining 1 mg or 1 μg of PF4ARin Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with 1/5 to1/10 the original amount of conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. 7 to 14 days later the animalsare bled and the serum is assayed for anti-PF4AR titer. Animals areboosted until the titer plateaus.

Another option is to employ combinatorial variable domain libraries andscreening methods to identify the desired anti-PF4AR antibodies.

Monoclonal antibodies are prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g. by fusion with myeloma cells or by Epstein-Barr (EB)-virustransformation and screening for clones expressing the desired antibody.

The monoclonal antibody preferably is specific for each target PF4ARpolypeptide, and will not cross-react with rabbit fMLP receptor (Thomaset al., J. Biol. Chem., supra), human fMLP receptor, human C5a receptorthe low affinity IL-8 receptor, or other members of the PF4AR family.Antibodies specific for the receptor of FIGS. 2, 4 or 5 are preferred.The antibody is selected to be either agonistic, antagonistic, or tohave no effect on the activity of a PF4 super-family member in bindingto or activating the receptor.

Murphy and Tiffany, supra, describe a receptor having a high degree ofhomology to the receptor of FIG. 2. Murphy and Tiffany characterizedtheir receptor in recombinant oocytes as being a receptor for IL-8 andhaving capability to bind MGSA, thus suggesting that it plays a minorrole in IL-8 and MGSA biological activity in vivo. The studies herein,however, have shown that the Murphy and Tiffany receptor exhibits IL-8affinity as high or higher than the receptor of FIG. 2 and that as wellit shows high affinity (about 1-10 nM) for MGSA. Thus, antagonism of theIL-8 and/or MGSA response of lymphoid cells will likely require thatboth receptors be inhibited or blocked. For example, one should selectan IL-8 antagonist antibody that binds to an epitope of the FIG. 2receptor that is shared by the Murphy and Tiffany receptor. This couldbe readily accomplished by routine screening methods. For example, thecandidate antibodies can be assayed for their ability to compete againstlabelled IL-8 for binding to cells bearing the FIG. 2 receptor, and thenthe same study conducted with cells bearing the Murphy and Tiffanyreceptor. Antibodies that inhibit IL-8 activation or binding to bothcells are then selected as therapeutic candidates. On the other hand,antibodies that can discriminate between the FIG. 2 and Murphy andTiffany receptors and bind only to one or the other are useful indiagnosis. The receptor of FIG. 2 binds MGSA poorly, in contrast to theMurphy and Tiffany receptor.

5. Uses of PF4AR its nucleic acid and its Antibodies

The nucleic acid encoding the PF4AR may be used as a diagnostic fortissue specific typing. For example, such procedures as in situhybridization, and northern and Southern blotting, and PCR analysis maybe used to determine whether DNA and/or RNA encoding the PF4AR arepresent in the cell type(s) being evaluated. These receptors typicallyare diagnostic of PBL or monocytic cells.

Isolated PF4AR polypeptide may be used in quantitative diagnostic assaysas a standard or control against which samples e.g. from PBL ormonocytic cells, containing unknown quantities of PF4AR may be compared.Recombinant cells which express the IL-8 receptor can be used in assaysfor PF4A ligands in the same fashion as for example neutrophils are usedin IL-8 assays. The PF4AR polypeptides, fragments or cells (as such, orderivatized) also can be used as immunogens in the production ofantibodies to PF4AR, for the purification of such antibodies fromascites or recombinant cell culture media or for use as competitiveanatagonists for superfamily ligands, e.g. IL-8.

The PF4AR are useful in screening for amino acid sequence or othervariants of PF4 superfamily members. For example, a bank of candidateIL-8 amino acid sequence variants are prepared by site directedmutagenesis. These are incubated in competition with labelled nativeIL-8 for cells bearing the IL-8 receptor of FIG. 2 in order identifyagonist or antagonist IL-8 variants. Binding or cell activation aresuitable assay endpoints. Alternatively, the receptor is recovered incell-free form and binding of IL-8 and candidate variants assayed.

PF4AR antibodies are useful in diagnostic assays for PF4AR expression inspecific cells or tissues wherein the antibodies are labeled in the samefashion as the PF4AR described above and/or are immobilized on aninsoluble matrix. PF4AR antibodies also are useful for the affinitypurification of the PF4AR from recombinant cell culture or naturalsources. The PF4AR antibodies that do not detectably cross-react withother PF4ARs can be used to purify each PF4AR free from other homologousreceptors. PF4AR antibodies that are PF4 antagonists are useful asanti-inflammatory agents or in the treatment of other PF4superfamily-mediated disorders.

Suitable diagnostic assays for the PF4AR and its antibodies are wellknown per se. Such assays include competitive and sandwich assays, andsteric inhibition assays. Competitive and sandwich methods employ aphase-separation step as an integral part of the method while stericinhibition assays are conducted in a single reaction mixture.Fundamentally, the same procedures are used for the assay of the PF4ARand for substances that bind the PF4AR, although certain methods will befavored depending upon the molecular weight of the substance beingassayed. Therefore, the substance to be tested is referred to herein asan analyte, irrespective of its status otherwise as an antigen orantibody, and proteins that bind to the analyte are denominated bindingpartners, whether they be antibodies, cell surface receptors, orantigens.

Analytical methods for the PF4AR or its antibodies all use one or moreof the following reagents: labeled analyte analogue, immobilized analyteanalogue, labeled binding partner, immobilized binding partner andsteric conjugates. The labeled reagents also are known as "tracers." Thelabel used (and this is also useful to label PF4AR nucleic acid for useas a probe) is any detectable functionality that does not interfere withthe binding of analyte and its binding partner. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores suchas rare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412(1982). Preferred labels herein are enzymes such as horseradishperoxidase and alkaline phosphatase.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al., "Methodsfor the Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay," in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.Such binding methods are suitable for use with PF4AR or its antibodies,all of which are proteinaceous.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a "homogeneous" assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte the presence of the anti-analyte modifies the enzyme activity.In this case, the PF4AR or its immunologically active fragments areconjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with anti-PF4AR so thatbinding of the anti-PF4AR inhibits or potentiates the enzyme activity ofthe label. This method per se is widely practiced under the name ofEMIT. Steric conjugates are used in steric hindrance methods forhomogeneous assay. These conjugates are synthesized by covalentlylinking a low-molecular-weight hapten to a small analyte so thatantibody to hapten substantially is unable to bind the conjugate at thesame time as anti-analyte. Under this assay procedure the analytepresent in the test sample will bind anti-analyte, thereby allowinganti-hapten to bind the conjugate, resulting in a change in thecharacter of the conjugate hapten, e.g., a change in fluorescence whenthe hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of PF4ARor PF4AR antibodies. In sequential sandwich assays an immobilizedbinding partner is used to adsorb test sample analyte, the test sampleis removed as by washing, the bound analyte is used to adsorb labeledbinding partner, and bound material is then separated from residualtracer. The amount of bound tracer is directly proportional to testsample analyte. In "simultaneous" sandwich assays the test sample is notseparated before adding the labeled binding partner. A sequentialsandwich assay using an anti-PF4AR monoclonal antibody as one antibodyand a polyclonal anti-PF4AR antibody as the other is useful in testingsamples for PF4AR activity.

The foregoing are merely exemplary diagnostic assays for PF4AR andantibodies. Other methods now or hereafter developed for thedetermination of these analytes are included within the scope hereof,including the bioassays described above.

The polypeptides set forth in FIGS. 4 and 5 are believed to representreceptors for different and as yet undetermined members of the PF4superfamily (which includes both the C-C and CXC subfamilies). Like theIL-8 receptor of FIG. 2 they are members of the G-protein-coupledsuperfamily and bear greater similarity to the IL-8 receptor than otherreceptors. In preliminary experiments, recombinant cells bearing thesereceptors do not respond to Rantes, MCP1, IL-8 or MGSA, although theymay ultimately be shown to bind other members of the PF4 superfamily orpresently unknown ligands. However, whether or not the FIGS. 4 or 5polypeptides bind to members of the PF4 superfamily, the polypeptidesare useful for preparing antibodies for diagnostic use in determiningthe tissue distribution of the receptors and thus as animmunohistochemical diagnostic for such tissues, in particular as adiagnostic for monocytic cells or PBLs since it is known that such cellsexpress the receptors of FIGS. 4 and 5. Of course, once the PF4superfamily members are identified which bind to these receptors thenthe receptors can be used to diagnose the presence of the identifiedmembers or for their purification in specific affinity procedures. TheDNA in FIGS. 4 and 5 also is useful in diagnostics for the presence ofDNA or RNA encoding the IL-8 receptor when low stringency conditions areemployed.

All references cited in this specification are expressly incorporated byreference.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1

To obtain the clone pRK5B.il8r1.1, a cDNA (Gubler and Hoffman, Gene, 25:263-269 1983!) library of 1,000,000 clones was constructed from humanneutrophil mRNA (Chirgwin et al., Biochem., 18: 5294-5299 1979!) in thevector pRK5B using BstXI linkers. The cDNA is produced in blunted form.

Hemi-kinase bstXI linkers are ligated to the cDNA, and the linkersligated into the PRK5B vector that had been bstXI digested,phosphatased, and the long vector fragment isolated. PRK5B is aderivative of PRK5 (EP 307,247) that contains a cytomegalovirus promoterfollowed by a 5' intron, bstXI cloning site, and an SV40 earlypolyadenylation signal, although it will be understood that anymammalian cell expression vector will be satisfactory, 58 pools of 2500clones each were transfected into COS-7 cells by electroporation(Gearing et al., supra) of 20 μg of DNA into 3,750,000 cells After 2days of growth on 150-mm dishes in medium (50:50: :Ham's F12:DMEM)containing 10% fetal calf serum, ¹²⁵ I-IL-8 binding was performed.Purified human 72 amino acid IL-8 made in E. coli (Hebert et al., J.Immunology, 145: 3033-3040 1990!) was labeled by the lactoperoxidasemethod (Morrison and Bayse, Biochem., 9: 2995-3000 1970!) to about 1100Ci/mmol and was at least 85% bindable. Dishes were rinsed twice withphosphate-buffered saline, and binding was performed with 8 ml per dishof growth medium containing 2.5% fetal calf serum and about 0.5 nM ¹²⁵I-IL-8. After 2 hr at 37° C., the plates were rinsed three times withphosphate-buffered saline, the bottoms cut out (Pacholczyk et al.,BioTechnicues, 9: 556-558 1990!), and autoradiographed. Each positivepool of 2500 cDNA clones was subsequently partitioned into pools of 800clones, and each of these was transfected and assayed. Each positivepool in turn was subdivided through pools of 185, 30 and finally asingle clone(s) until single positive clones were identified to obtainthe pure isolate. Since only a portion of each pool was used fortransfection it was unnecessary to rescue clones from transformants.

Binding competition was performed with electroporated COS-7 cells after1 day of expression in 6-well dishes (about 175,000 cells/dish). Bindingwas performed with radioiodinated wild type IL-8 in binding medium Ca²⁺and Mg²⁺ -free Hanks buffered with 25 nM Hepes and supplemented with0.5% bovine serum albumin BSA!) at 4° C. for about 2 hr. Wells were thenwashed, the cells harvested with trypsin, and counted. No specificbinding was found in parallel wells containing cells transfected withDNA from the vector pRK5B. Neutrophil binding was performed as described(Pacholczyk et al., supra) but for 2 hr at 4° C.

EXAMPLE 2

Existing λgt10 cDNA libraries from the human cell line, HL60, and fromhuman peripheral blood lymphocytes were screened at low stringency witha probe from the coding region of the cloned high-affinity human IL-8receptor (FIG. 2). The probe was the 874 bp PstI/NcoI fragment of thereceptor containing the coding region for amino acids 23-314.Hybridization was in 20% formamide, 4×SSC, 50 mM sodium phosphatebuffer, pH 7, 0.2 g/l sonicated salmon sperm DNA, 5×Denhardts, 10%dextran sulfate, at 420C with a wash at 1×SSC, 0.1% SDS at 50° C. Anumber of duplicate spots of varying intensity (about 60) were picked,plaque purified, subcloned into plasmid vectors, and sequenced. Nucleicacid sequencing began with the selection of spots of greatest intensity.Sufficient sequence was obtained for a given spot (phage) to determinewhether or not evidence of structural or sequence homology with the IL-8receptor existed. If it did, then the remainder of the gene was obtained(if necessary) and sequenced in its entirety.

To avoid sequences all hybridizing clone the sequence was then used toprobe the parental collection of IL-8 receptor DNA hybridizing clonesunder high stringency conditions in order to identify and discard otherspots containing the same hybridizing gene. This technique was highlyeffective in reducing the sequencing burden. For example, one clone wasrepresented by about one third of the initial 60 clones, and on thisresult alone the negative screen was able to reduce considering the workinvolved in sequencing the clones.

From this screen, two new gene sequences were found that are clearlyrelated to the IL-8 receptor. The coding region for one new gene wassplit between two clones (8rr.20 and 8rr.15). The combined sequence ofthis gene (8rr.20.15) is shown in FIG. 4. The complete coding region forthe second gene is found on clone 8rr.9 (FIG. 5). The predicted aminoacid sequence of 8rr.20.15 is 34% identical with both the high and lowaffinity IL-8 receptor sequences. The sequence of 8rr.9 is 36% and 38%identical with the high and low affinity IL-8 receptor sequences,respectively (Holmes et al., Science, 253: 1278 1991! and Murphy andTiffany, supra). The amino acid sequence of 8rr.20.15 and 8rr.9 are 31%identical. Use of this probe under low stringency conditions did notproduce detectable hybridization to the fMLP receptor genes that wereexpected to be found in these libraries.

EXAMPLE 3

Monoclonal antibodies to IL-8 type A receptor were generated byimmunizing mice with synthetic peptides corresponding to variousextracellular domains of IL8R-A or with stably transfected cellsexpressing IL8R-A, respectively. Blocking and non-blocking monoclonalantibodies were identified and their binding sites were mapped to theN-terminal region of IL8R-A. Details are provided below:

Experimental Protocol

Recombinant human IL-8 (rHuIL-8) was produced in E. coli and purified asdescribed in Hebert et al., supra.

For generating IL8R-A-bearing cells, human fetal kidney 293 cells wereco-transfected with pRK5B.il8r1.1 (Holmes et al., supra) orpRK5.8rr27-1.1 (Lee et al., J. Biol. Chem., 267: 16283-16287 1992!) andpSVENeoBal6 (Seeburg et al., Nature, 312: 71-75 1984!) plasmids in a10:1 molar ratio, using a CaPO₄ precipitation method as described inGorman in DNA Cloning: A Practical Approach, ed., Glover, D. M. (IRL:Oxford, 1984), Vol. 2, pp. 143-165. Transfected cells were selected inF12/DMEM (50:50) media containing 10% containing 10% fetal calf serum(FCS), 2 mM L-glutamine, 100 μg/ml penicillin, and 100 μg/mlstreptomycin. Forty G418-resistant clonal lines were isolated from thepRK5B.il8r1.1 transfection. The IL8R-A-bearing transfected cells wereselected by their ability to bind to ¹²⁵ I-IL-8, and clone 293-71 wasused for further study. Thirty G418-resistant clonal lines were isolatedfrom the pRK5.8rr.27-1.1 transfection. After the ¹²⁵ I-IL-8 bindingexperiment, clone 36 was selected for further study.

Mutants were prepared by the method of Kunkel, Proc. Natl. Acad. Sci.USA, 82: 488 (1985) using the dut- ung- strain of E. coli CJ236 and apUC-derived vector containing a cDNA insert coding for the IL-8 receptorA: pRKSB.IL-8r1.1. Holmes et al., supra. After verification of themutant DNA sequence with the Sequenase™ version 2.0 kit (U.S.Biochemical Corp.), the mutated plasmid preparations were purified withthe Qiagen™ plasmid maxi kit (Qiagen Inc., Chatsworth, Calif.) and usedto transfect 293 cells by the calcium phosphate precipitate method ofGorman, supra. The cell cultures were incubated for seven hours in thepresence of mutant or wild-type DNA precipitate (10 μg DNA/100 mm dish).The precipitate was then removed and the cells were cultured for anadditional 17 hours prior to fluorescence-activated cell sorter (FACS)analysis.

Peptides were synthesized via solid-phase methodology (Barany andMerrifield, in "the peptides," 2: 1-284, Gross and Meienhofer, eds,Academic Press: New York, 1980!) on either an ABI model 430 peptidesynthesizer using tert-butyloxycarbonyl (t-BOC) chemistry or a Milligenmodel 9050 and ABI model 431 peptide synthesizer usingfluorenylmethyloxycarbonyl (FMOC) chemistry. Crude peptides werepurified by high-pressure liquid chromatography (HPLC) and analyzed viamass spectrometry. Peptides were conjugated to soybean trypsin inhibitorusing m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS)(Pierce Co., Rockford, Ill.).

BALB/c mice were immunized intraperitoneally with 10 μg of syntheticpeptides covering various portions of extracellular domains of IL8R-Aconjugated to horse serum albumin or 10⁶ cells/100 μl of 293-71transfected cells, resuspended in MPL/TDM (Ribi Immunochem. ResearchInc., Hamilton, Mont.) and boosted nine times with the same amount ofpeptides or 16 times with transfected cells. Three days after the finalboost with the antigen, spleen cells were fused with mouse myelomaP3X63Ag8U.1 (Yelton et al., Curr. Top. Microbiol. Immunol., 81: 1-71978!), a non-secreting clone of the myeloma P3X63Ag8 (Kohler andMilstein, Nature, 256: 495 1975!) using 35% polyethylene glycol asdescribed by Laskov et al., Cell. Immunol., 55: 251-264 (1980). Ten daysafter the fusion, culture supernatants were screened for the presence ofmonoclonal antibodies to IL8R-A by an ELISA or FACS.

Nunc™ brand 96-well immunoplates (Flow Lab, McLean, Va.) were coatedwith 50 μl/well of 2 μg/mL IL8R-A synthetic peptide in phosphatebuffered saline (PBS) overnight at 4° C. The remaining steps werecarried out at room temperature as described by Kim et al., J. Imm.Methods., 156: 9 (1992). The isotypes of monoclonal antibodies weredetermined by coating the plates with IL8R-A peptides overnight, blockedwith 0.2% BSA, incubated with culture supernatants, followed by theaddition of a predetermined amount of isotype-specific alkalinephosphatase-conjugated goat anti-mouse Ig (Fisher Biotech, Pittsburgh,Pa.). The level of conjugated antibodies that bound to the plate wasdetermined by the addition of p-nitrophenyl phosphate substrate incarbonate buffer containing 1 mM of MgCl₂ (Sigma 104 phosphatesubstrate, Sigma, St. Louis, Mo.) . The color reaction was measured at405 nm with an ELISA plate reader (Titertrek multiscan, Flow Lab,McLean, Va.).

Human neutrophils were prepared by using Mono-Poly Resolving medium(M-PRM) (Flow Lab, McLean, Va.) according to the vendor's direction.Neutrophils or transfected cells were washed twice in the cell sorterbuffer (CSB, PBS containing 1% FCS and 0.02% NaN₃) at 300×g for 5minutes. Twenty-five μl of cells (4×10⁶ cells/ml) were added into a96-well U-bottom microtiter plate, mixed with 100 μl of culturesupernatant or purified monoclonal antibodies, and incubated for 30 min.on ice. After washing, cells were incubated with 100 μl ofFITC-conjugated goat-anti-mouse IgG antibodies for 30 min. at 4° C.Cells were washed twice in CSB and resuspended in 150 μl of CSB andanalyzed by a FCAScan™ assay (Becton-Dickinson).

The affinity of the monoclonal antibodies was determined by competitiveinhibition of the binding of ¹²⁵ I-monoclonal antibody to293-71-transfected cells with various concentrations of unlabeledmonoclonal antibodies. ¹²⁵ I-monoclonal antibodies were prepared byusing chloramine-T as described by Lee et al., supra. The specificactivities of monoclonal antibodies 6C8, 6E9, 2A4, and 9H1 were 0.68Ci/M, 0.74 Ci/M, 0.814 Ci/M, and 0.922 Ci/M, respectively. Fifty μl of293-17 (4×10⁶ cells/ml) resuspended in Hank's buffered saline solution(HBSS) containing 0.5% BSA, were briefly incubated with 100 μl of afixed concentration of ¹²⁵ I-monoclonal antibody plus 50 μl of variousconcentrations of unlabeled monoclonal antibody for one hour at 4° C.The unbound ¹²⁵ I-monoclonal antibody was removed by centrifugation ofthe mixture over 0.5 ml of PBS containing 20% sucrose and 0.5% BSA at1,500 rpm for five minutes. The ¹²⁵ I-monoclonal antibody bound to thecell pellet was determined using a gamma counter. The affinity of eachmonoclonal antibody was determined by using Scatplot™ analysis. Munsonand Rodbard, Anal. Biochem., 107: 220 (1980).

The blocking ability of the monoclonal antibodies was determined bymeasuring their effects on the interaction of IL-8 with IL8R-A. Fifty μlof human neutrophils or transfected cells (4×10⁶ cells/ml) resuspendedin the HBSS medium containing 0.5% BSA and 25 mM HEPES buffer wereincubated with 50 μl of monoclonal antibodies plus variousconcentrations of MGSA (0-0.5 nM) for one hour at 4° C. Cells werewashed twice in the HBSS medium and resuspended to be 1×10⁶ cells/ml.One hundred μl of cells were incubated with 100 μl of ¹²⁵ I-IL-8 (1Ci/M) for one hour at 4° C. and the unbound ¹²⁵ I-IL-8 was removed using20% sucrose as described above. The ¹²⁵ I-IL-8 bound to the cell pelletswas counted using a gamma counter.

Results

General Characteristics of the antibodies.

For generation of monoclonal antibodies to IL8R-A, mice were immunizedeither with synthetic peptides corresponding to various extracellulardomains of IL8R-A or with stably transfected cells expressing IL8R-A.

For the first approach, eight peptides were synthesized covering theextracellular domains of IL8R-A, residues 2-19 and 12-31 located withinthe N-terminal portion of IL8R-A, 99-110 within the first loop, 176-186and 187-203 within the second loop, and 265-277 and 277-291 within thethird loop as shown in FIG. 6. All of these peptides induced high-titerantibodies to each peptide in mice. However, only peptide 2-19 producedpolyclonal antibodies that were able to recognize human neutrophils aswell as 293-71-transfected cells expressing IL8R-A. The mice immunizedwith peptide 2-19 were used to generate 36 hybridomas secretingmonoclonal antibodies to peptide 2-19. Only two of these monoclonalantibodies (4C8 and 6E9) were able to recognize IL8R-A on293-transfected cells and were selected for further characterization.

In a second approach, mice were immunized with 293-71 cells producingIL8R-A (293-71). Positive antibody titers were detected only after the16th immunization. These mice were used to obtain more than 60 positivehybridomas that secreted monoclonal antibodies recognizing IL8R-A on293-71 cells, as determined by FACS. Two out of 60 monoclonalantibodies, 2A4 and 9H1, were able to inhibit the binding of ¹²⁵ I-IL-8to its receptors and were chosen for further evaluation.

These four hybridomas secrete IgGl immunoglobulin isotype (Table 2).Monoclonal antibodies 2A4 and 9H1, generated using transfected cells,have higher affinities than monoclonal antibodies 4C8 and 6E9, generatedusing peptides (Table 2). All four monoclonal antibodies were able tobind to human neutrophils and 293-71-transfected cells but not to 293parent cells, as determined by FACS analysis (FIG. 7). Thus, it wasconcluded that all these monoclonal antibodies were capable ofrecognizing native IL8R-A.

                  TABLE 2    ______________________________________                     FACS Analysis                                  Kd    Mab    Immunogen Isotype   293-71                                     Neutrophil                                              (nM)    ______________________________________    4C8    Peptide 2-19                     IgG1      +     +        3.26    6E9    Peptide 2-19                     IgG1      +     +        17.26    2A4    293-71    IgG1      +     +        0.44    9H1    293-71    IgG1      +     +        0.088    ______________________________________

Cross reactivities to other related receptors.

It has been shown that IL-8 specific receptor, IL8R-A, shares 77% aminoacid identity with IL8R-B, the common IL-8/MGSA receptor. Fordetermining whether monoclonal antibodies generated to IL8R-A couldrecognize IL8R-B, 293 cells transfected with IL8R-B were stained andanalyzed by FACS (FIG. 7). All four monoclonal antibodies stained theIL8R-A 293-transfected cells, but not the IL8R-B-transfected 293 cells.The inability of these monoclonal antibodies to bind to 293 cellsexpressing IL8R-B was not due to the lack of receptor expression, sincethe same level of ¹²⁵ I-IL-8 was bound to IL8R-A expressing transfectedcells, as well as to the IL8R-B expressing transfected cells.

Inhibition of IL-8 binding to IL8R-A.

The effect of the monoclonal antibodies on the binding of ¹²⁵ I-IL-8 tothe 293-71-transfected cells expressing IL8R-A (FIG. 8) wasinvestigated. Monoclonal antibodies 2A4 and 9H1 could completely blockthe ¹²⁵ I-IL-8 binding to the 293-71 cells, whereas monoclonalantibodies 4C8 and 6E9 showed very minimal effect.

It was further investigated whether monoclonal antibodies 2A4 and 9H1could block IL-8 binding onto human neutrophils in the presence ofvarious concentrations of MGSA, known to bind to IL8R-B. The addition of475 pM of MGSA inhibited approximately 50% of IL-8 binding to humanneutrophils (FIG. 8). In the presence of 475 pM of MGSA, monoclonalantibodies 2A4 and 9H1 could block up to 80% of the IL-8 binding tohuman neutrophils, while the control monoclonal antibody showed nofurther inhibition beyond that observed with MGSA alone. Thus, it wasconcluded that monoclonal antibodies 2A4 and 9H1 could block the bindingof IL-8 to IL8R-A on human neutrophils.

Epitope mapping of the IL8R-A specific monoclonal antibodies.

The epitopes recognized by these monoclonal antibodies were mapped byinvestigating the binding of these antibodies to synthetic peptides byELISA (FIGS. 9A and 9B) and alanine mutants of IL8R-A by FACS (Table 3).Surprisingly, both blocking and non-blocking monoclonal antibodies boundto the N-terminal peptide consisting of amino acids 2-19, but did notbind to other peptides covering different portions of extracellularloops of the receptor (FIG. 9A).

The binding of monoclonal antibodies to shorter peptides consisting ofresidues 1-11 and 1-14 in ELISA is shown in FIG. 9B. All four monoclonalantibodies bound well to peptide 1-14. However, the binding of theblocking monoclonal antibodies, 2A4 and 9H1, to peptide 1-11 was only22% and 60% of the binding to peptide 2-19, respectively. In contrast,the binding of the non-blocking monoclonal antibodies 4C8 and 6E9 topeptide 1-11 was approximately 80% and 95% of the binding to peptide2-19, respectively. From these results, it was concluded that epitopesof monoclonal antibodies 4C8 and 6E9 are localized within amino acids2-11, while those of monoclonal antibodies 2A4 and 9H1 are localizedwithin amino acids 2-14.

                  TABLE 3    ______________________________________    Flow Cytometry Analysis of Antibodies with Cells    Expressing Mutant IL-8 Type A Receptor    Amino Acid              Change     FACS Analysis    Position  From    To     2A4    9H1  4C8    6E9    ______________________________________    6         D       A      -      -    +      +    11        D       A      ++     ++   +      +    13-14     DD      A      ++     ++   +      ++    24-26     DED     AAA    ++     ++   ++     +    ______________________________________     FACS Result: - (mean FL channel 0-10), + (Mean FL channel 10-100, ++ (Mea     FL channel 100-1000).     A (Alanine), D (Aspartic acid), K (Lysine), E (Glutamic Acid)

The binding epitopes of these monoclonal antibodies were furtherinvestigated by analyzing their binding to IL8R-A mutants by FACS (Table3). Neither monoclonal antibody 2A4 nor 9H1 could bind to the IL8R-Amutant when the aspartic acid at position 6 was substituted withalanine, suggesting Asp6 plays an important role in the binding of theseblocking antibodies. This result further suggests that the conformationof the N-terminal end of the IL8R-A may play a role in the binding ofthese blocking monoclonal antibodies.

Conclusion

IL-8 is a potent neutrophil chemotactic factor and has been implicatedas a key mediator of neutrophil influx in many inflammatory diseases.IL-8 has been detected in many biological fluids from patients with avariety of acute and chronic inflammatory diseases such as arthritis,emphysema, cystic fibrosis, ulcerative colitis, chronic bronchitis, andbronchiectasis. Koch et al., J. Immunol., 147: 2187-2195 (1991); Koch etal., Science, 258: 1798-1801 (1992); Terkeltaub et al., Arthr. andRheum., 34: 894 (1991); Mahida et al., Clin. Science, 82: 273-275(1992); Broaddus et al., Am. Rev. Rest., 146: 825 (1992). In vitroneutralization of the IL-8 in these biological fluids significantlyreduces the overall chemotactic activity in the fluid. Further, the roleof IL-8 is implicated in inflammatory conditions where the cellularinfitltrate is predominantly neutrophil-rich, including gout, rheumatoidarthritis, adult respiratory distress syndrome, emphysema, glomerularnephritis, myocardial infarction, inflammatory bowel disease, andasthma. Lindley et al., Adv. Exp. Med., 305: 147-156 (1991). However, towhat extent IL-8 contributes to inflammation in vivo has yet to bedetermined.

Various monoclonal antibodies directed against IL-8 have been defined,including those reported by Sticherling et al., J. Immunol., 143:1628-1634 (1989).

It is anticipated that an antagonist to IL8R-A will be more effective inblocking the effect of IL-8 function in vivo than an antagonist to IL-8for the following reasons: IL-8 is relatively long-lasting and resistantto proteases, possibly making it difficult to block IL-8 completely invivo, the IL8R-A expression is dynamically regulated by the liganditself, and the rapid recycling of IL-8 receptors may be essential forthe chemotactic response of neutrophils and tissue damage duringinflammation, caused by activated neutrophils. Thus, monoclonalantibodies to IL8R-A expressed on human neutrophils were generated withthe ultimate goal of identifying neutralizing monoclonal antibodies thatcould be tested as receptor antagonists of various inflammation in invivo models.

The antibody responses to different extracellular portions of IL8R-Awere induced using several synthetic peptides covering the extracellulardomains of IL8R-A or IL8R-A-transfected cells as immunogens. Bothmethods of immunization allowed generation of monoclonal antibodiesrecognizing IL8R-A on human neutrophils (FIG. 6). It was much harder togenerate monoclonal antibodies specific for IL8R-A by using transfectedcells as an immunogen; however, monoclonal antibodies generated usingtransfected cells tended to have higher affinities to IL8R-A and showedblocking activities.

Polyclonal antibody responses were able to be induced to all sevenpeptides covering the extracellular domains of IL8R-A, residues 2-19,1231, 99-110, 176-186, 187-203, 265-277, and 277-291. However, only thepolyclonal antibodies to peptide 2-19 recognize IL8R-A on cells. Thissuggests that the N-terminal amino acids may be the most immunogenic orthat the most likely immunogenic sites have conformational epitopes.

A 77% sequence identity between IL8R-A and IL8R-B was reported by Holmeset al., supra; however, the antibodies herein were specific for IL8R-A.IL8R-A does not recognize IL-1, TNF-α, MCAF, fMLP, C5α, PAF, and LTB4,but does recognize two other members of the C-X-C family, namely, MGSAand NAP-2. Holmes et al., supra. A recent study shows that bothreceptors bind IL-8 equally well with a high affinity but differ intheir affinity to MGSA. Murphy and Tiffany, supra. Monoclonal antibodies2A4 and 9H1 block 100% of the IL-8 binding to transfected 293 cells,35-40% of the IL-8 binding to human neutrophils, and approximately 80%of IL-8 binding to human neutrophils in the presence of MGSA, whichpresumably blocked IL-8 binding to IL8R-B. Thus, the blocking monoclonalantibodies herein interfere with the interaction between IL-8 andIL8R-A, but not with the interaction between IL-8 and IL8R-B. Bothmonoclonal antibodies 4C8 and 6E9 showed no blocking activities in IL-8binding to both transfected cells and human neutrophils.

The finding that both blocking and non-blocking monoclonal antibodiesbind to the N-terminal amino acid residues 1-14 (FIG. 9A) is consistentwith the report that the N-terminal amino acid sequence of IL8R-A playsan important role in interacting with IL-8. Gayle et al., J. Biol.Chem., 268: 7283-7289 (1993). Further epitope mapping analysis showedsome differences in the binding characteristics between blocking andnon-blocking monoclonal antibodies. The epitopes of blocking monoclonalantibodies 2A4 and 9H1 were mapped within residues 2-14 of the receptor,while those of non-blocking monoclonal antibodies 4C8 and 6E9 werewithin 2-11 amino acid residues. The binding study using various alaninemutants shows that the aspartic acid at position 6 plays an importantrole in the binding of the blocking monoclonal antibodies, but not ofthe non-blocking monoclonal antibodies. This suggests that thisnegatively charged aspartic acid may be located at (or near to) thebinding site of IL-8. It has been shown that positively charged IL-8residues E₄, L₅, R₆ are essential for IL-8 binding to its receptors onhuman neutrophils by alanine-scanning mutagenesis (Hebert et al., J.Biol. Chem., 266: 18989-18994 1991!) and synthesis of N-terminaltruncated variants (Clark-Lewis et al., J. Biol. Chem., 266: 23128-231341991!; Moser et al., J. Biol. Chem., 268: 7125-7128 1993!). Withoutbeing limited to any one theory, it is believed that the negativelycharged amino acid at position 6 in IL8R-A is in a direct or closeinteraction with the positively charged amino acids, E₄, L₅, R₆, inIL-8, so that the binding of blocking monoclonal antibodies isinfluenced by the tertiary structure of the receptor.

The N-terminal portion and the second extracellular portion of theIL8R-A appears to be highly glycosylated, especially within amino acids2-19 of the receptor, where there are six potential glycosylation sites(FIG. 6). However, all of the monoclonal antibodies herein, whethergenerated by immunization of synthetic peptides or of transfected cells,bound to the synthetic peptide covering amino acids 1-14. This suggeststhat carbohydrates may not play an important role in the binding ofthese monoclonal antibodies.

EXAMPLE 4

IL-8 was found to be present at high concentration and is the majorneutrophil chemotactic factor in sputum from patients with chronicbronchitis, bronchiectasis, and cystic fibrosis. FIG. 10 shows theconcentration of IL-8 in sputum from patients with chronic airwaydiseases and in sputum induced from healthy patients.

An ELISA is developed to measure rabbit IL-8 concentration using adouble monoclonal antibody technique where one antibody is used as thecoat and the other is the biotinylated detection antibody. In the assaybuffer the sensitivity is <12 pg/ml (linear range 12-1000 pg/ml) . In10% rabbit serum the sensitivity is 14 pg/ml (linear range 14-1000pg/ml).

One of the two antibodies 2A4 and 9H1 described above is injectedintravenously every two weeks in a dose of 1-15 mg/kg in patients havingeither asthma, chronic bronchitis, bronchiectesis, rheumatoid arthritis,or ulcerative colitis. For treating an acute indication, adultrespiratory distress syndrome, a dose of 10-100 mg/kg of either one ofthe antibodies is injected a single time intravenously. It would beexpected that the anti-IL8R-A antibodies that block IL8R-A activity(MAbs 2A4 and 9H1) would be effective in reducing the inflammationassociated with each of the disorders described above. The antibodiesare also expected to be efficacious in treating human pleurisy,vasculitis, alveolitis, and pneumonia.

Deposit of Materials

The following hybridomas have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________    Cell Lines  ATCC Accession No.                              Deposit Date    ______________________________________    2A4         HB 11377      June 8, 1993    9H1         HB 11376      June 8, 1993    ______________________________________

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable deposit for 30 years fromthe date of deposit. These cell lines will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the cell lines to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the cell lines to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the depositedcell lines should be lost or destroyed when cultivated under suitableconditions, they will be promptly replaced on notification with aspecimen of the same cell line. Availability of the deposited cell linesis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines deposited,since the deposited embodiments are intended as illustrations of oneaspect of the invention and any cell lines that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 6    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1933 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #              50CAGTTA GATCAAACCA TTGCTGAAAC TGAAGAGGAC    #             100AGATCC ACAGATGTGG GATTTTGATG ATCTAAATTT    #             150CTGCAG ATGAAGATTA CAGCCCCTGT ATGCTAGAAA    #             200AAGTAT GTTGTGATCA TCGCCTATGC CCTAGTGTTC    #             250GGGAAA CTCCCTGGTG ATGCTGGTCA TCTTATACAG    #             300CCGTCA CTGATGTCTA CCTGCTGAAC CTGGCCTTGG    #             350GCCCTG ACCTTGCCCA TCTGGGCCGC CTCCAAGGTG    #             400TGGCAC ATTCCTGTGC AAGGTGGTCT CACTCCTGAA    #             450ACAGTG GCATCCTGCT GTTGGCCTGC ATCAGTGTGG    #             500ATTGTC CATGCCACAC GCACACTGAC CCAGAAGCGT    #             550TGTTTG TCTTGGCTGC TGGGGACTGT CTATGAATCT    #             600TCCTTT TCCGCCAGGC TTACCATCCA AACAATTCCA    #             650GAGGTC CTGGGAAATG ACACAGCAAA ATGGCGGATG    #             700GCCTCA CACCTTTGGC TTCATCGTGC CGCTGTTTGT    #             750ATGGAT TCACCCTGCG TACACTGTTT AAGGCCCACA    #             800CGAGCC ATGAGGGTCA TCTTTGCTGT CGTCCTCATC    #             850GCTGCC CTACAACCTG GTCCTGCTGG CAGACACCCT    #             900TGATCC AGGAGACCTG TGAGCGCCGC AACAACATCG    #             950GCCACT GAGATTCTGG GATTTCTCCA TAGCTGCCTC    #            1000CGCCTT CATCGGCCAA AATTTTCGCC ATGGATTCCT    #            1050TGCATG GCCTGGTCAG CAAGGAGTTC TTGGCACGTC    #            1100TACACT TCTTCGTCTG TCAATGTCTC TTCCAACCTC    #            1150GAAGGA ATATCTCTTC TCAGAAGGAA AGAATAACCA    #            1200TGTGTG GAAGGTGATC TGGCTCTGGA CAGGCACTAT    #            1250GACGCT ATAGGATGTG GGGAAGTTAG GAACTGGTGT    #            1300CCAACC TTCTGAGGAG CTGTTGAGGT ACCTCCAAGG    #            1350CTCCAT GGAAACGAAG CACCATCATT CCCGTTGAAC    #            1400CCACTA ACTGGCTAAT TAGCATGGCC ACATCTGAGC    #            1450TAGATG AGAGAACAGG GCTGAAGCTG TGTCCTCATG    #            1500TCGTTG ACCCTCACAG GAGCATCTCC TCAACTCTGA    #            1550GCCACC AAGCTGGTGG CTCTGTGTGC TCTGATCCGA    #            1600TTTTCC CATCTCAGGT GTGTTGCAGT GTCTGCTGGA    #            1650CACTGC CAAAACATCA ACCTGCCAGC TGGCCTTGTG    #            1700CATGTT CCCCTTGGGG GTGGTGGATG AACAAAGAGA    #            1750GCCAGA TCTATGCCAC AAGAACCCCC TTTACCCCCA    #            1800GACACA TGTGCTGGCC ACCTGCTGAG CCCCAAGTGG    #            1850CCCTTA GCCCTTCCCC TCTGCAGCTT CCAGGCTGGC    #            1900TCCCTA GAAAGCCATG TGCAGCCACC AGTCCATTGG    #       1933       AATA AAGCTTCTGT TCC    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 350 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Ser Asn Ile Thr Asp Pro Gln Met Trp As - #p Phe Asp Asp Leu    #                 15    - Asn Phe Thr Gly Met Pro Pro Ala Asp Glu As - #p Tyr Ser Pro Cys    #                 30    - Met Leu Glu Thr Glu Thr Leu Asn Lys Tyr Va - #l Val Ile Ile Ala    #                 45    - Tyr Ala Leu Val Phe Leu Leu Ser Leu Leu Gl - #y Asn Ser Leu Val    #                 60    - Met Leu Val Ile Leu Tyr Ser Arg Val Gly Ar - #g Ser Val Thr Asp    #                 75    - Val Tyr Leu Leu Asn Leu Ala Leu Ala Asp Le - #u Leu Phe Ala Leu    #                 90    - Thr Leu Pro Ile Trp Ala Ala Ser Lys Val As - #n Gly Trp Ile Phe    #                105    - Gly Thr Phe Leu Cys Lys Val Val Ser Leu Le - #u Lys Glu Val Asn    #               120    - Phe Tyr Ser Gly Ile Leu Leu Leu Ala Cys Il - #e Ser Val Asp Arg    #               135    - Tyr Leu Ala Ile Val His Ala Thr Arg Thr Le - #u Thr Gln Lys Arg    #               150    - His Leu Val Lys Phe Val Cys Leu Gly Cys Tr - #p Gly Leu Ser Met    #               165    - Asn Leu Ser Leu Pro Phe Phe Leu Phe Arg Gl - #n Ala Tyr His Pro    #               180    - Asn Asn Ser Ser Pro Val Cys Tyr Glu Val Le - #u Gly Asn Asp Thr    #               195    - Ala Lys Trp Arg Met Val Leu Arg Ile Leu Pr - #o His Thr Phe Gly    #               210    - Phe Ile Val Pro Leu Phe Val Met Leu Phe Cy - #s Tyr Gly Phe Thr    #               225    - Leu Arg Thr Leu Phe Lys Ala His Met Gly Gl - #n Lys His Arg Ala    #               240    - Met Arg Val Ile Phe Ala Val Val Leu Ile Ph - #e Leu Leu Cys Trp    #               255    - Leu Pro Tyr Asn Leu Val Leu Leu Ala Asp Th - #r Leu Met Arg Thr    #               270    - Gln Val Ile Gln Glu Thr Cys Glu Arg Arg As - #n Asn Ile Gly Arg    #               285    - Ala Leu Asp Ala Thr Glu Ile Leu Gly Phe Le - #u His Ser Cys Leu    #               300    - Asn Pro Ile Ile Tyr Ala Phe Ile Gly Gln As - #n Phe Arg His Gly    #               315    - Phe Leu Lys Ile Leu Ala Met His Gly Leu Va - #l Ser Lys Glu Phe    #               330    - Leu Ala Arg His Arg Val Thr Ser Tyr Thr Se - #r Ser Ser Val Asn    #               345    - Val Ser Ser Asn Leu                    350    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1737 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #              50TGGCGG CGCGGCGCAA AGTGACGCCG AGGGCCTGAG    #             100CCGCAT CTGGAGAACC AGCGGTTACC ATGGAGGGGA    #             150TCAGAT AACTACACCG AGGAAATGGG CTCAGGGGAC    #             200GGAACC CTGTTTCCGT GAAGAAAATG CTAATTTCAA    #             250CCACCA TCTACTCCAT CATCTTCTTA ACTGGCATTG    #             300GTCATC CTGGTCATGG GTTACCAGAA GAAACTGAGA    #             350GTACAG GCTGCACCTG TCAGTGGCCG ACCTCCTCTT    #             400CCTTCT GGGCAGTTGA TGCCGTGGCA AACTGGTACT    #             450TGCAAG GCAGTCCATG TCATCTACAC AGTCAACCTC    #             500CATCCT GGCCTTCATC AGTCTGGACC GCTACCTGGC    #             550CCAACA GTCAGAGGCC AAGGAAGCTG TTGGCTGAAA    #             600GGCGTC TGGATCCCTG CCCTCCTGCT GACTATTCCC    #             650CAACGT CAGTGAGGCA GATGACAGAT ATATCTGTGA    #             700ATGACT TGTGGGTGGT TGTGTTCCAG TTTCAGCACA    #             750ATCCTG CCTGGTATTG TCATCCTGTC CTGCTATTGC    #             800GCTGTC ACACTCCAAG GGCCACCAGA AGCGCAAGGC    #             850TCATCC TCATCCTGGC TTTCTTCGCC TGTTGGCTGC    #             900ATCAGC ATCGACTCCT TCATCCTCCT GGAAATCATC    #             950GTTTGA GAACACTGTG CACAAGTGGA TTTCCATCAC    #            1000TCTTCC ACTGTTGTCT GAACCCCATC CTCTATGCTT    #            1050TTTAAA ACCTCTGCCC AGCACGCACT CACCTCTGTG    #            1100CCTCAA GATCCTCTCC AAAGGAAAGC GAGGTGGACA    #            1150CTGAGT CTGAGTCTTC AAGTTTTCAC TCCAGCTAAC    #            1200CTTTTT TTTATACGAT AAATAACTTT TTTTTAAGTT    #            1250TATAAA AGACTGACCA ATATTGTACA GTTTTTATTG    #            1300GTCTTG TGTTTCTTTA GTTTTTGTGA AGTTTAATTG    #            1350ATTTTT TTTGTTTCAT ATTGATGTGT GTCTAGGCAG    #            1400TTCTTA GTTGCTGTAT GTCTCGTGGT AGGACTGTAG    #            1450CATTCC AGAGCGTGTA GTGAATCACG TAAAGCTAGA    #            1500GTTTAT GCATAGATAA TCTCTCCATT CCCGTGGAAC    #            1550TAAGAC GTGATTTTGC TGTAGAAGAT GGCACTTATA    #            1600TGGTAT AGAAATGCTG GTTTTTCAGT TTTCAGGAGT    #            1650ACCTAC AGTGTACAGT CTTGTATTAA GTTGTTAATA    #            1700ACTTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA    #    1737          GCCG CCAGCACACT GGAATTC    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 352 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Met Glu Gly Ile Ser Ile Tyr Thr Ser Asp As - #n Tyr Thr Glu Glu    #                 15    - Met Gly Ser Gly Asp Tyr Asp Ser Met Lys Gl - #u Pro Cys Phe Arg    #                 30    - Glu Glu Asn Ala Asn Phe Asn Lys Ile Phe Le - #u Pro Thr Ile Tyr    #                 45    - Ser Ile Ile Phe Leu Thr Gly Ile Val Gly As - #n Gly Leu Val Ile    #                 60    - Leu Val Met Gly Tyr Gln Lys Lys Leu Arg Se - #r Met Thr Asp Lys    #                 75    - Tyr Arg Leu His Leu Ser Val Ala Asp Leu Le - #u Phe Val Ile Thr    #                 90    - Leu Pro Phe Trp Ala Val Asp Ala Val Ala As - #n Trp Tyr Phe Gly    #                105    - Asn Phe Leu Cys Lys Ala Val His Val Ile Ty - #r Thr Val Asn Leu    #               120    - Tyr Ser Ser Val Leu Ile Leu Ala Phe Ile Se - #r Leu Asp Arg Tyr    #               135    - Leu Ala Ile Val His Ala Thr Asn Ser Gln Ar - #g Pro Arg Lys Leu    #               150    - Leu Ala Glu Lys Val Val Tyr Val Gly Val Tr - #p Ile Pro Ala Leu    #               165    - Leu Leu Thr Ile Pro Asp Phe Ile Phe Ala As - #n Val Ser Glu Ala    #               180    - Asp Asp Arg Tyr Ile Cys Asp Arg Phe Tyr Pr - #o Asn Asp Leu Trp    #               195    - Val Val Val Phe Gln Phe Gln His Ile Met Va - #l Gly Leu Ile Leu    #               210    - Pro Gly Ile Val Ile Leu Ser Cys Tyr Cys Il - #e Ile Ile Ser Lys    #               225    - Leu Ser His Ser Lys Gly His Gln Lys Arg Ly - #s Ala Leu Lys Thr    #               240    - Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cy - #s Trp Leu Pro Tyr    #               255    - Tyr Ile Gly Ile Ser Ile Asp Ser Phe Ile Le - #u Leu Glu Ile Ile    #               270    - Lys Gln Gly Cys Glu Phe Glu Asn Thr Val Hi - #s Lys Trp Ile Ser    #               285    - Ile Thr Glu Ala Leu Ala Phe Phe His Cys Cy - #s Leu Asn Pro Ile    #               300    - Leu Tyr Ala Phe Leu Gly Ala Lys Phe Lys Th - #r Ser Ala Gln His    #               315    - Ala Leu Thr Ser Val Ser Arg Gly Ser Ser Le - #u Lys Ile Leu Ser    #               330    - Lys Gly Lys Arg Gly Gly His Ser Ser Val Se - #r Thr Glu Ser Glu    #               345    - Ser Ser Ser Phe His Ser Ser    #   352         350    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1679 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    #NO:5:  (xi) SEQUENCE DESCRIPTION: SEQ ID    #              50TGGCGG CCGCCCAGTG TGCTGGCGGC GGCAGTTGAG    #             100TTATGA GTGCCTGCAA GAGTGGCAGC CTGGAGTAGA    #             150TGGAGT CAAAAGACCT GAGTTCAAGT CCCAGCTCTG    #             200TGGGAT CTCGGAAAAG ACCCAGTGAA AAAAAAAAAA    #             250TGAGGC AGGTCGCGGC CCTACTGCCT CAGGAGACGA    #             300GCTTAA ATTTGCAGCT GACGGCTGCC ACCTCTCTAG    #             350GAGCCT CTCAACATAA GACAGTGACC AGTCTGGTGA    #             400AGCCAT GAACTACCCG CTAACGCTGG AAATGGACCT    #             450ACCTGT TCTGGGAACT GGACAGATTG GACAACTATA    #             500GTGGAA AATCATCTCT GCCCTGCCAC AGAGGGGCCC    #             550CAAGGC CGTGTTCGTG CCCGTGGCCT ACAGCCTCAT    #             600TGATCG GCAACGTCCT GGTGCTGGTG ATCCTGGAGC    #             650CGCAGT TCCACGGAGA CCTTCCTGTT CCACCTGGCC    #             700GCTGGT CTTCATCTTG CCCTTTGCCG TGGCCGAGGG    #             750TCCTGG GGACCTTCCT CTGCAAAACT GTGATTGCCC    #             800TTCTAC TGCAGCAGCC TGCTCCTGGC CTGCATCGCC    #             850GGCCAT TGTCCACGCC GTCCATGCCT ACCGCCACCG    #             900TCCACA TCACCTGTGG GACCATCTGG CTGGTGGGCT    #             950CCAGAG ATTCTCTTCG CCAAAGTCAG CCAAGGCCAT    #            1000GCCACG TTGCACCTTC TCCCAAGAGA ACCAAGCAGA    #            1050TCACCT CCCGATTCCT CTACCATGTG GCGGGATTCC    #            1100GTGATG GGCTGGTGCT ACGTGGGGGT AGTGCACAGG    #            1150GCGGCG CCCTCAGCGG CAGAAGGCAG TCAGGGTGGC    #            1200GCATCT TCTTCCTCTG CTGGTCACCC TACCACATCG    #            1250ACCCTG GCGAGGCTGA AGGCCGTGGA CAATACCTGC    #            1300TCTCCC CGTGGCCATC ACCATGTGTG AGTTCCTGGG    #            1350GCCTCA ACCCCATGCT CTACACTTTC GCCGGCGTGA    #            1400CTGTCG CGGCTCCTGA CGAAGCTGGG CTGTACCGGC    #            1450CCAGCT CTTCCCTAGC TGGCGCAGGA GCAGTCTCTC    #            1500CCACCT CTCTCACCAC GTTCTAGGTC CCAGTGTCCC    #            1550TTTCCT TGGGGCAGGC AGTGATGCTG GATGCTCCTT    #            1600GATCCT AAGGGCTCAC CGTGGCTAAG AGTGTCCTAG    #            1650GGGGTA GCTAGAGGAA CCAACCCCCA TTTCTAGAAC    #          1679    CACA CTGGAATTC    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 372 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Met Asn Tyr Pro Leu Thr Leu Glu Met Asp Le - #u Glu Asn Leu Glu    #                 15    - Asp Leu Phe Trp Glu Leu Asp Arg Leu Asp As - #n Tyr Asn Asp Thr    #                 30    - Ser Leu Val Glu Asn His Leu Cys Pro Ala Th - #r Glu Gly Pro Leu    #                 45    - Met Ala Ser Phe Lys Ala Val Phe Val Pro Va - #l Ala Tyr Ser Leu    #                 60    - Ile Phe Leu Leu Gly Val Ile Gly Asn Val Le - #u Val Leu Val Ile    #                 75    - Leu Glu Arg His Arg Gln Thr Arg Ser Ser Th - #r Glu Thr Phe Leu    #                 90    - Phe His Leu Ala Val Ala Asp Leu Leu Leu Va - #l Phe Ile Leu Pro    #                105    - Phe Ala Val Ala Glu Gly Ser Val Gly Trp Va - #l Leu Gly Thr Phe    #               120    - Leu Cys Lys Thr Val Ile Ala Leu His Lys Va - #l Asn Phe Tyr Cys    #               135    - Ser Ser Leu Leu Leu Ala Cys Ile Ala Val As - #p Arg Tyr Leu Ala    #               150    - Ile Val His Ala Val His Ala Tyr Arg His Ar - #g Arg Leu Leu Ser    #               165    - Ile His Ile Thr Cys Gly Thr Ile Trp Leu Va - #l Gly Phe Leu Leu    #               180    - Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Se - #r Gln Gly His His    #               195    - Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gl - #n Glu Asn Gln Ala    #               210    - Glu Thr His Ala Trp Phe Thr Ser Arg Phe Le - #u Tyr His Val Ala    #               225    - Gly Phe Leu Leu Pro Met Leu Val Met Gly Tr - #p Cys Tyr Val Gly    #               240    - Val Val His Arg Leu Arg Gln Ala Gln Arg Ar - #g Pro Gln Arg Gln    #               255    - Lys Ala Val Arg Val Ala Ile Leu Val Thr Se - #r Ile Phe Phe Leu    #               270    - Cys Trp Ser Pro Tyr His Ile Val Ile Phe Le - #u Asp Thr Leu Ala    #               285    - Arg Leu Lys Ala Val Asp Asn Thr Cys Lys Le - #u Asn Gly Ser Leu    #               300    - Pro Val Ala Ile Thr Met Cys Glu Phe Leu Gl - #y Leu Ala His Cys    #               315    - Cys Leu Asn Pro Met Leu Tyr Thr Phe Ala Gl - #y Val Lys Phe Arg    #               330    - Ser Asp Leu Ser Arg Leu Leu Thr Lys Leu Gl - #y Cys Thr Gly Pro    #               345    - Ala Ser Leu Cys Gln Leu Phe Pro Ser Trp Ar - #g Arg Ser Ser Leu    #               360    - Ser Glu Ser Glu Asn Ala Thr Ser Leu Thr Th - #r Phe    #   372         370    __________________________________________________________________________

What is claimed is:
 1. A method for determining the presence or absenceof the nucleic acid of SEQ ID NO:3 or its complement in a sample,comprising the steps of:(a) selecting a probe comprising at least 20contiguous nucleotides selected from the nucleic acid sequence of SEQ IDNO:3 or its complement, (b) hybridizing the probe to any nucleic acid ofSEQ ID NO:3 or its complement present in a sample to form aprobe/nucleic acid complex, (c) detecting the presence or absence of theprobe/nucleic acid complex in the sample, and (d) determining thepresence or absence of the nucleic acid of SEQ ID NO:3 or its complementin the sample based on the result of step (c).